Nurr1:rxr activating compounds for simultaneous treatment of symptoms and pathology of parkinson&#39;s disease

ABSTRACT

The invention provides a series of substituted aryl pyrimidine compounds and the use of these compounds as therapeutics to treat or prevent neurodegenerative disorders, including Parkinson&#39;s disease. Compounds of the invention are also able to treat the symptoms of such diseases and therefore represent a new treatment modality for ameliorating chronic and acute conditions. The compounds of the invention are capable of selectively potentiating the activity of the Nurr1:RXRα heterodimer, and are able to treat diseases or conditions associated with aberrant Nurr1:RXRα function. The invention further provides methods for treating neurodegenerative disorders by administration of Nurr1:RXRα activating agents.

BACKGROUND OF THE INVENTION Neurodegenerative Diseases

Neurodegenerative diseases are characterized by progressive dysfunctionof the nervous system and frequently are associated with gradualdeterioration and death of affected neurons. Generally, these diseasesare associated with aging and include Alzheimer's disease, Huntington'sdisease, Parkinson's disease, Steele-Richardson-Olszewski syndrome,Multiple-system atrophy, and amyotrophic lateral sclerosis. Consideringthat overall life expectancy is rising, age-associated neurodegenerativediseases, such as Parkinson's and Alzheimer's disease, constitute thefastest growing group of diseases worldwide and represent an enormoussocietal and financial burden on the western world. Among theneurodegenerative diseases, Alzheimer's disease is the most common, andParkinson's disease affects nearly seven to ten million people worldwideof any age, but it is most common in people older than 65 years.

Parkinson's disease affects movement, leading to progressive loss ofmuscle control, leading to trembling of the limbs and head while atrest, stiffness, slowness, and impaired balance. As symptoms worsen, itmay become difficult to walk, talk, and complete simple tasks. Thedisease is accompanied or even preceded by non-motor symptoms thatinclude autonomic dysfunction, neuropsychiatric symptoms affecting moodand cognition, as well as sensory and sleep disturbances.

The symptoms of Parkinson's disease are associated with the greatlyreduced activity and number of dopaminergic neurons of the substantianigra in the midbrain. Lack of dopamine and loss of dopaminergic neuronprojections to the striatum lead to activity alterations in the basalganglia that regulate movement. Dopamine depletion in other non-striataldopamine pathways is thought to explain some of the neuropsychiatricpathology associated with the disease.

The current treatment for PD is symptomatic and is based on replenishingthe dopamine deficiency through the administration of the dopaminemetabolic precursor L-3,4-dihydroxyphenylalanine (L-DOPA), which wasdiscovered in the 1960's. L-DOPA restores temporarily DA levels andalleviates DA dependent motor and some non-motor symptoms. ProlongedL-DOPA treatment leads to dyskinesias (abnormal involuntary movements),which abolishes its beneficial effect (Prashanth 2011). Additionally,L-DOPA treatment does not stop the neuron degeneration, and, on thecontrary it might be toxic, increasing neuronal degeneration.

Only a limited number of compounds have been tested for neuroprotectionin clinical trials of PD patients because it is not possible to measurethe number of dopaminergic neurons in vivo (Henchcliffe 2011). Thus, ithas been thought that a realistic goal for Parkinson's therapies is tominimize symptoms, rather than to see if we can extend the life of braincells. In addition, the effects of PD medications that patients arereceiving complicate the evaluation of the extent of nerve cell loss.Ideally, a compound that improves both the symptoms of PD but alsoprevents or diminishes neuronal loss could become a way to cure thedisease.

Nurr1

Nurr1 (NR4A2), an orphan nuclear receptor that is required for midbraindopaminergic neuron development, regulates the expression of genesinvolved in dopamine biosynthesis, Neuropilin1, VIP, BDNF and inresistance to oxidative stress (Hermanson 2006, Luo 2007, Volpicelli2007, Sousa 2007). While Nurr1−/− mice die shortly after birth and failto develop midbrain dopaminergic neurons, Nurr1+/− mice appear normalbut with reduced striatal dopamine levels. Nurr1 expression in midbraindopaminergic neurons continues throughout life. During aging, Nurr1expression levels decrease and decrease even further in the substantianigra of PD patients. Importantly, six Nurr1 mutations have been foundin familial (2) and sporadic (4) PD cases linking the function of thegene with PD. The two familial mutations were detected in all affectedindividuals of each family in ten different families. Five of themutations have been mapped to the first untranslated exon of Nurr1 andresult in decreased steady state mRNA levels (Le 2003, Hering 2004,Healy 2006, Grimes 2006, Sleiman 2009). The sixth mutation is in thecoding region affecting a phosphorylation site and results in decreasedtranscriptional activity of the protein.

As a transcription factor Nurr1 binds to DNA as monomer, as a homodimeror as a heterodimer with RXR (Retinoid X Receptor) α and γ, nuclearreceptors that form heterodimers with several other members of thenuclear receptor family. RXRα expression is ubiquitous, while expressionof RXRγ is detected primarily in the brain and particularly in thestriatum, the hypothalamus and the pituitary.

Retinoid X Receptors (RXRs)

RXRs are nuclear receptors that are activated by 9-cis retinoic acid.RXRs have been shown to be important in development and differentiationand to be involved in many processes, including metabolism. There arethree retinoic X receptors (RXR): RXR-α, RXR-13, and RXR-γ. All threeare capable of forming heterodimers with a variety of nuclear receptorsincluding RAR, CAR, FXR, LXR, PPAR, PXR, RAR, TR, NR4A2 (nurr1), NR4A1and VDR. The activity of these heterodimers can be modulated by RXRligands.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a compound of formula(I):

or a pharmaceutical acceptable salt thereof, where n is 0 to 2, p is 0to 2, X is N(R₆) or O;Cy is a phenyl ring or a heteroaromatic 5- or 6-membered ring containingbetween 1 and 4 heteroatoms;each R₁ is independently selected from the group consisting of halogen,cyanate, isocyanate, thiocyanate, isothiocyanate, selenocyanate,isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano, nitro,hydroxy, acyl, mercapto, carboxyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR₇,—SR₇, and —N(R₈)R₇;each R₂ is independently selected from the group consisting of halogen,cyanate, isocyanate, thiocyanate, isothiocyanate, selenocyanate,isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano, nitro,hydroxy, acyl, mercapto, carboxyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR₇,—SR₇, and —N(R₈)R₇;R₃ is selected from the group consisting of hydrogen, halogen, cyanate,isocyanate, thiocyanate, isothiocyanate, selenocyanate,isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano, nitro,hydroxy, acyl, mercapto, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, OR₇, —SR₇,—N(R₈)R₇, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted cycloalkyl and optionally substitutedheterocyclyl, or R₃ and R₆, when present, combine with the atoms towhich they are bound to form an optionally substituted 5-memberedheteroaryl or heterocyclyl;R₄ is selected from the group consisting of hydrogen, halogen,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted aryl-alkyl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted heteroaryl-alkyl, optionally substitutedheterocyclolalkyl, OR₇, and —N(R₈)R₇;R₅ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, and optionallysubstituted aryl;R₆ is selected from the group consisting of hydrogen and optionallysubstituted alkyl;each R₇ is independently selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted aryl, optionally substituted aryl-alkyl, and optionallysubstituted heterocyclyl; andeach R₈ is independently selected from the group consisting of hydrogenand optionally substituted alkyl.

In certain embodiments, the compound is not a compound selected from thegroup consisting of:

-   i. ethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   ii. methyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   iii. 4-(6-methyl-2-phenyl-5-propylpyrimidin-4-ylamino)benzoic acid;-   iv. 4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic    acid;-   v. 4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   vi.    4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ylamino)benzoic    acid;-   vii. 4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   viii. 4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid;-   ix. 4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yloxy)benzoic    acid;-   x. 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   xi. ethyl 4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   xii. methyl 4-(2,6-diphenylpyrimidin-4-yloxy)benzoate;-   xiii. 3-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   xiv. ethyl 4-(2,6-diphenylpyrimidin-4-ylamino)benzoate;-   xv. methyl 4-(2-(4-bromophenyl)-6-methylpyrimidin-4-yloxy)benzoate;-   xvi. 4-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-yloxy)benzoic acid;-   xvii. ethyl    4-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xviii. methyl    4-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xix. methyl    3-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xx.    4-(5-carboxy-2-methoxyanilino)-2-(pyrazin-2-yl)-6-trifluoromethylpyrimidine;-   xxi.    4-(5-carboxy-2-hydroxyanilino)-2-(pyridin-3-yl)-6-(trifluoromethyl)pyrimidine;-   xxii.    4-[2-methyl-5-(carboxymethylester)anilino]-2-(pyridin-3-yl)-6-(trifluoromethyl)pyrimidine;-   xxiii.    4-(5-carboxy-2-methoxyanilino)-2-(pyridin-3-yl)-6-(trifluoromethyl)pyrimidine;    and-   xxiv. 2-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid.

In certain embodiments, the compound is not a compound selected from thegroup consisting of:

-   i. ethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   ii. methyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   iii. 4-(6-methyl-2-phenyl-5-propylpyrimidin-4-ylamino)benzoic acid;-   iv. 4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic    acid;-   v. 4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   vi.    4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ylamino)benzoic    acid;-   vii. 4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   viii. 4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid;-   ix. 4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yloxy)benzoic    acid;-   x. 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   xi. ethyl 4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   xii. methyl 4-(2,6-diphenylpyrimidin-4-yloxy)benzoate;-   xiii. 3-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   xiv. ethyl 4-(2,6-diphenylpyrimidin-4-ylamino)benzoate;-   xv. methyl 4-(2-(4-bromophenyl)-6-methylpyrimidin-4-yloxy)benzoate;-   xvi. 4-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-yloxy)benzoic acid;-   xvii. ethyl    4-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xviii. methyl    4-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xix. methyl    3-(2-(2-hydroxyphenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xx.    4-(5-carboxy-2-methoxyanilino)-2-(pyrazin-2-yl)-6-trifluoromethylpyrimidine;-   xxi.    4-(5-carboxy-2-hydroxyanilino)-2-(pyridin-3-yl)-6-(trifluoromethyl)pyrimidine;-   xxii.    4-[2-methyl-5-(carboxymethylester)anilino]-2-(pyridin-3-yl)-6-(trifluoromethyl)pyrimidine;-   xxiii.    4-(5-carboxy-2-methoxyanilino)-2-(pyridin-3-yl)-6-(trifluoromethyl)pyrimidine;-   xxiv. 2-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid;-   xxv. 2′,3′-dihydroxypropyl    2-(6-methyl-2-(3-trifluoromethylphenyl)pyrimidin-4-ylamino)benzoate;-   xxvi. methyl    2-(6-methyl-2-(3-trifluoromethylphenyl)pyrimidin-4-ylamino)benzoate;-   xxvii. 2′,2′-dimethyl-1′,3′-dioxolan-4′-ylmethyl    2-(6-methyl-2-(3-trifluoromethylphenyl)pyrimidin-4-ylamino)benzoate;-   xxviii. ethyl    4-(2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxix. methyl    2-(2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxx. 2′,2′-dimethyl-1′,3′-dioxolan-4′-ylmethyl    2-(2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxxi. 2′,3′-dihydroxypropyl    2-(2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxxii. ethyl    4-(2-(3-fluorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxxiii. methyl    2-(2-(3-fluorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxxiv. 2′,2′-dimethyl-1′,3′-dioxolan-4′-ylmethyl    2-(2-(3-fluorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxxv. 2′,3′-dihydroxypropyl    2-(2-(3-fluorophenyl)-6-methylpyrimidin-4-ylamino)benzoate;-   xxxvi. ethyl    4-(6-methyl-2-(3-trifluoromethylphenyl)pyrimidin-4-ylamino)benzoate;-   xxxvii. ethyl    4-(6-methyl-2-(3,4-methylenedioxyphenyl)pyrimidin-4-ylamino)benzoate;-   xxxviii. methyl    2-(6-methyl-2-(3,4-methylenedioxyphenyl)pyrimidin-4-ylamino)benzoate;-   xxxix. 2′,2′-dimethyl-1′,3′-dioxolan-4′-ylmethyl    2-(6-methyl-2-(3,4-methylenedioxyphenyl)pyrimidin-4-ylamino)benzoate;-   xl. 2′,3′-dihydroxypropyl    2-(6-methyl-2-(3,4-methylenedioxyphenyl)pyrimidin-4-ylamino)benzoate;-   xli. ethyl    4-(6-methyl-2-(3,4,5-trimethoxyphenyl)pyrimidin-4-ylamino)benzoate;-   xlii. methyl    2-(6-methyl-2-(3,4,5-trimethoxyphenyl)pyrimidin-4-ylamino)benzoate;-   xliii. 2′,2′-dimethyl-1′,3′-dioxolan-4′-ylmethyl    2-(6-methyl-2-(3,4,5-trimethoxyphenyl)pyrimidin-4-ylamino)benzoate;-   xliv. 2-[(6-methyl-2-morpholin-4-ylpyrimidin-4-yl)amino]benzoic    acid;-   xlv.    2-{[6-methyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-yl]amino}benzoic    acid; and-   xlvi. 2-(6-chloro-2-phenylpyrimidin-4-ylamino)benzoic acid.

In some embodiments, R₃ is not hydrogen. In some embodiments, Cy is aphenyl ring, R₃ is optionally substituted alkyl or optionallysubstituted alkenyl, R₄ is optionally substituted methyl (e.g.,halomethyl), and R₅ is hydrogen or optionally substituted alkyl. Forinstance, in some embodiments, Cy is a phenyl ring, R₃ is alkyl oralkenyl, R₄ is methyl or trifluoromethyl, and R₅ is hydrogen or alkyl.

In particular embodiments of this aspect, n in formula (I) above is 0.In alternative embodiments, n is 0 or 1, and p is 0 or 1. In certainembodiments, X is NR₆, optionally wherein R₄ is haloalkyl, such astrifluoromethyl.

In other embodiments, R₃ and R₆ combine with the atoms to which they arebound to form an optionally substituted 5-membered heteroaryl orheterocyclyl. For example, the compound is a compound of the formula(1e):

where R₁₀ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaryl-alkyl, optionally substituted heteroaryl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; andR₁₁ is selected from the group consisting of optionally substitutedalkyl, optionally substituted aryl, optionally substituted aryl-alkyl,optionally substituted heteroaryl, optionally substituted cycloalkyl,and optionally substituted heterocyclyl.

Additional embodiments of the invention include compounds of formula(1b):

or a free base thereof, where L is selected from the group consisting of—O—, —O—R₉—O—, and —O—R₉—, wherein R₉ is an optionally substitutedalkylene, optionally substituted alkenylene, or optionally substitutedalkynylene chain.

In other embodiments, the compound is a compound of formula (1f):

or a salt thereof,where Y is —O—R₉—, wherein R₉ is an optionally substituted alkylene,optionally substituted alkenylene, or optionally substituted alkynylenechain.

Embodiments of the invention further include compounds of formula (1g):

or a salt thereof, Additional embodiments of the above aspects of theinvention include compounds of formula (1h):

or a salt thereof,

In particular embodiments of the invention, X in the above formulas isO.

In an additional aspect, the compound is selected from the groupconsisting of:

-   a)    4-((5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-yl)(methyl)amino)benzoic    acid;-   b) 4-(4,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzoic    acid;-   c)    3-bromo-4-(4,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzoic    acid;-   d) 4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoic    acid;-   e)    4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-2-fluorobenzoic    acid;-   f)    4-(5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoic    acid;-   g)    (E)-4-(2-(4-chlorophenyl)-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoic    acid;-   h)    4-((5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yl)(methyl)amino)benzoic    acid;-   i)    4-(5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoic    acid;-   j)    (E)-4-(2-phenyl-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoic    acid;-   k)    4-((2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yl)(methyl)amino)benzoic    acid;-   l) pivaloyloxymethyl    4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate;-   m) 4-((6-methyl-2-phenylpyrimidin-4-yl)(propyl)amino)benzoic acid;-   n)    (S)-2-(4-(4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoyloxy)phenyl)-1-carboxyethanaminium    chloride; and-   o)    (S)-2-(4-(3-(4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoyloxy)propoxy)phenyl)-1-carboxyethanaminium    chloride;    or a salt or free base thereof.

In embodiments of any of the above described aspects of the invention,the compound may be incorporated into a pharmaceutical composition incombination with a pharmaceutically acceptable excipient. In particularembodiments, the pharmaceutical composition may contain an additionaltherapeutically active compound. For example, in certain cases, theadditional therapeutically active compound is selected from the groupconsisting of levodopa (L-dihydroxyphenylalanine), L-aromatic amino aciddecarboxylase (AADC) inhibitors, and catechol O-methyl transferase(COMT) inhibitors.

In addition, the present invention provides a method of treating aneurodegenerative disorder in a human by administration to the human ofan effective amount of a compound or pharmaceutical compositiondescribed above. The invention further provides a method of alleviatingor preventing one or more symptoms of a neurodegenerative disorder in ahuman by administration to the human of an effective amount of acompound or pharmaceutical composition described above. In particularcases, the symptom is selected from the group consisting of bradykinesiaand dyskinesias. In other embodiments, the symptom is selected from thegroup consisting of anxiety and depression. The symptom mayalternatively be selected from the group consisting of learningdifficulties, memory disorders, and attention deficit disorder. In othercases, the symptom may be selected from the group consisting of insomniaand katalepsy.

The present invention additional relates to a method of treating adisorder in a human patient in which Nurr1 activity is reduced orimpaired relative to a healthy human by administration to the human ofan effective amount of a compound or pharmaceutical compositiondescribed above.

In embodiments of any of the above-described methods of the invention,the disorder that can be treated by administration to the human of aneffective amount of a compound or pharmaceutical composition describedherein is Parkinson's disease.

In particular embodiments of the methods described above, the effectiveamount is sufficient to halt or prevent degeneration of neurons. Incertain cases, these neurons are dopaminergic midbrain neurons.Additionally or alternatively, the effective amount is sufficient toincrease the rate of dopamine biosynthesis. In certain cases, theeffective amount is sufficient to increases the rate of transcription ortranslation of a polynucleotide encoding an enzyme involved in dopaminebiosynthesis. For example, in particular embodiment, the enzyme isselected from the group consisting of tyrosine hydroxylase (TH),aromatic amino acid decarboxylase (AADC), and phenylalanine hydroxylase(PheOH). In alternative embodiments, the effective amount is sufficientto increases the rate of tetrahydrobiopterin (BH4) biosynthesis. Incertain cases, the effective amount is sufficient to increase the rateof transcription or translation of a polynucleotide encoding an enzymeinvolved in BH4 biosynthesis. For instance, in particular embodiments,the enzyme is GTP cyclohydrolase 1 (GCH1). In alternative embodiments,the effective amount is sufficient to increase the rate of serotonin(5-HT) biosynthesis. In particular cases, the effective amount issufficient to increases the rate of transcription or translation of apolynucleotide encoding an enzyme involved in 5-HT biosynthesis. Forinstance, in certain embodiments, the enzyme is selected from the groupconsisting of tryptophan hydroxylase (TPH) and amino acid decarboxylase(DDC).

In certain embodiments of the above-described methods, the compoundselectively activates a Nurr1:RXRα heterodimer over one or more otherRXRα-containing dimers. Additionally or alternatively, the compound mayincrease the rate of dopamine biosynthesis and serotonin biosynthesis.

The present invention additionally provides a method of increasing therate of dopamine biosynthesis in a human by administration to the humanof an effective amount of a compound or pharmaceutical compositiondescribed above. In certain cases, the administration increases the rateof serotonin biosynthesis.

The invention further relates to a method of treating aneurodegenerative disorder in a human by administration to the human ofan effective amount of a compound capable of activating Nurr1. Theinvention additionally features the compound of any of the aspects orembodiments described above for use in treating a neurodegenerativedisorder (e.g., Parkinson's disease) in a human patient. Compounds ofany of the aspects or embodiments described above can be used foralleviating or preventing one or more symptoms of a neurodegenerativedisorder in a human patient, such as bradykinesia, dyskinesias, anxiety,depression, learning difficulties, memory disorders, attention deficitdisorder, insomnia, and katalepsy. The invention also features thecompound of any of the aspects or embodiments described above for use intreating a disorder in a human patient in which Nurr1 activity isreduced or impaired relative to a healthy human. The inventionadditionally features the compound of any of the aspects or embodimentsdescribed above for use in increasing the rate of dopamine biosynthesisand/or serotonin biosynthesis in a human.

In another aspect, the invention features a kit containing a compound orpharmaceutical composition as described herein. The kit may contain apackage insert, for example, instructing a user of the kit to perform amethod described herein, such as to administer a compound orpharmaceutical composition of the invention to a patient (e.g., a humanpatient) to treat a neurodegenerative disorder, such as Parkinson'sdisease, to alleviate or prevent one or more symptoms of aneurodegenerative disorder, such as bradykinesia, dyskinesias, anxiety,depression, learning difficulties, memory disorders, attention deficitdisorder, insomnia, and katalepsy, to treat a disorder in a humanpatient in which Nurr1 activity is reduced or impaired relative to ahealthy human, and/or to increase the rate of dopamine biosynthesisand/or serotonin biosynthesis in a human patient.

Definitions

As used herein, “acyl” refers to an —C(O)R moiety, wherein R ishydrogen, optionally substituted alkyl, optionally substituted alkenyl,or optional substituted alkynyl.

As used herein, “alkoxy” refers to a —O—R moiety, where R is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl group.

As used herein, alkyl, alkenyl, and alkynyl carbon chains, unlessotherwise specified, contain 1 to 6 carbons and can be straight orbranched. Unless otherwise specified, alkenyl carbon chains of 2 to 6carbons contain 1 to 2 double bonds. Unless otherwise specified, alkynylcarbon chains of 2 to 6 carbons contain 1 to 2 triple bonds. Exemplaryalkyl, alkenyl and alkynyl groups herein include, but are not limitedto, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl,tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl, allyl(propenyl)and propargyl(propynyl).

As used herein, “alkylamino” refers to a —N(R₁)R₂ moiety, where R₁ andR₂ are, independently, optionally substituted alkyl, optionallysubstituted alkenyl, or optionally substituted alkynyl groups.

As used herein, the “amelioration” of a symptom of a particular disorderby administration of a particular compound or pharmaceutical compositionrefers to any lessening of the symptom, whether permanent or temporary,that can be attributed to or associated with administration of thecompound or pharmaceutical composition.

As used herein, the term “amino acid” refers to α-amino acids that areracemic or of either the D- or L-configuration. The designation “d”preceding an amino acid designation (e.g., dAla, dSer, dVal, etc.)refers to the D-isomer of the amino acid. The designation “dl” precedingan amino acid designation (e.g., dlPip) refers to a mixture of the L-and D-isomers of the amino acid. It is to be understood that the chiralcenters of the compounds provided herein may undergo epimerization invivo. Amino acids may be of either the L- or D-form. The configurationfor naturally occurring amino acid residues is generally L. When notspecified, the residue is the L form.

As used herein, “aryl” refers to aromatic monocyclic or multicyclicgroups containing from 6 to 19 carbon atoms. Aryl groups include, butare not limited to, unsubstituted or substituted fluorenyl,unsubstituted or substituted phenyl, and unsubstituted or substitutednaphthyl.

As used herein, “aryl-alkyl” refers to an alkyl group in which one ofthe hydrogen atoms of the alkyl is replaced by an aryl group.

As used herein, “azido” refers to a —N═N═N moiety.

As used herein, the term “carboxyl” refers to a —COOH moiety.

As used herein, “cyanate” refers to a —O—C≡N moiety.

As used herein, “cyano” refers to a —C≡N moiety.

As used herein, the term “cycloalkyl” refers to a saturated mono- ormulti-cyclic ring system of from 3 to 6 carbon atoms. The ring systemsof the cycloalkyl groups may be composed of one ring or two or morerings which may be joined together in a fused, bridged orspiro-connected fashion.

As used herein, “EC₅₀” refers to a dosage, concentration, or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced,provoked, or potentiated by the particular test compound.

As used herein, the suffix “-ene” refers to a divalent version of theparent group.

As used herein, the term “halogen” refers to fluorine, chlorine,bromine, and/or iodine moieties.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by one or more halogen atoms.Such groups include, but are not limited to, chloromethyl,trifluoromethyl, and 1-chloro-2-fluoroethyl.

As used herein, “heteroaryl-alkyl” refers to an alkyl group in which oneof the hydrogen atoms of the alkyl is replaced by a heteroaryl group.

As used herein, the term “heteroaryl” refers to a monocyclic ormulticyclic aromatic ring system, in certain embodiments, of 5 to 15members where one or more (e.g., from 1 to 4) of the atoms in the ringsystem is a heteroatom (O, N, or S). A heteroaryl group may beoptionally fused to another ring, such as an optionally substitutedphenyl ring. Heteroaryl groups include, without limitation, furyl,imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl,thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl,and isoquinolinyl.

As used herein, the term “heteroatom” refers to an element other thancarbon, including, without limitation, nitrogen, oxygen, or sulfur.

As used herein, the terms “heterocyclyl” refer to a monocyclic ormulticyclic non-aromatic ring system, in one embodiment of 3 to 10members, in another embodiment of 4 to 7 members, in a furtherembodiment of 5 to 6 members, where one or more (e.g., from 1 to 4) ofthe atoms in the ring system is a heteroatom (e.g., O, N, or S). Inembodiments where the heteroatom(s) is(are) nitrogen, the nitrogen maybe optionally substituted with alkyl, alkenyl, alkynyl, aryl,heteroaryl, aryl-alkyl, heteroaryl-alkyl, cycloalkyl, heterocyclyl,cycloalkylalkyl, heterocyclylalkyl, acyl, or guanidino moieties, or thenitrogen may be quaternized to form an ammonium group containingsubstituents selected from those listed above.

As used herein, heterocyclyl-alkyl refers to an alkyl group in which oneof the hydrogen atoms of the alkyl is replaced by a heterocyclyl group

As used herein, “hydroxy” refers to a —OH moiety.

As used herein, “isocyanate” refers to a —N═C═O moiety.

As used herein, “isoselenocyanate” refers to a —N═C═Se moiety.

As used herein, “isothiocyanate” refers to a —N═C═S moiety.

As used herein, “mercapto” refers to a —SR moiety in which R is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl, andaryl.

As used herein, “nitro” refers to a —NO₂ moiety.

As used herein, “optionally substituted alkyl,” “optionally substitutedalkenyl,” “optionally substituted alkynyl,” “optionally substitutedcycloalkyl,” “optionally substituted cycloalkenyl,” “optionallysubstituted cycloalkynyl,” “optionally substituted aryl,” “optionallysubstituted heteroaryl” and “optionally substituted heterocyclyl” referto alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,aryl, heteroaryl, and heterocyclyl groups, respectively, that, whensubstituted, are independently substituted with one or more substituentsselected from the group consisting of a halogen, cyanate, isocyanate,thiocyanate, isothiocyanate, selenocyanate, isoselenocyanate, alkoxy,trifluoromethoxy, haloalkyl, alkylamino, azido, cyano, nitro, hydroxy,acyl, mercapto, carboxyl, ester, alkyl group, alkenyl group, alkynylgroup, aryl group, aryl-alkyl group, heteroaryl group, heteroaryl-alkylgroup, cycloalkyl group, heterocyclyl group, —OR₇, —SR₇, and —N(R₈)R₇,where R₇ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, aryl, aryl-alkyl, heteroaryl, heteroaryl-alkyl,cycloalkyl, and heterocyclyl, or R7 and R8, when present, may combinewith the atom to which they are bound to form a heteroaryl orheterocyclyl ring; and R₈ is selected from the group consisting ofhydrogen and alkyl.

As used herein, the term “prevention” refers to the prophylactictreatment of a subject who is at risk of developing a condition (e.g., aneurodegenerative disorder) resulting in a decrease in the probabilitythat the subject will develop the condition.

As used herein, “selenocyanate” refers to a —Se—C≡N moiety.

As used herein, “thiocyanate” refers to a —S—C≡N moiety.

As used herein, the term “treatment” refers to any manner in which oneor more of the symptoms of a disease or disorder are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein, such as a use fortreating a disease or disorder as described herein.

As used herein, the term “trifluoromethoxy” refers to a —OCF₃ moiety.

Where the number of any given substituent is not specified (e.g.,haloalkyl), there may be one or more substituents present. For example,“haloalkyl” may include one or more of the same or different halogens.As another example, “C₁₋₃ alkoxyphenyl” may include one or more of thesame or different alkoxy groups independently containing one, two, orthree carbons.

When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms of carbonyl-containing compounds are alsointended to be included.

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or may be stereoisomeric ordiastereomeric mixtures. As such, one of skill in the art will recognizethat administration of a compound in its (R) form is equivalent, forcompounds that undergo epimerization in vivo, to administration of thecompound in its (S) form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1G show various biochemical properties of compound 3. FIG. 1Ashows a schematic representation of the components used in a luciferaseassay as described herein. FIG. 1B shows the results of a screen ofselect compounds of the invention in a luciferase reporter assay toevaluate the Nurr1-activating properties of these compounds. XCT, aknown Nurr1 activator, is used as a positive control. FIG. 1C shows theresults of a luciferase assay to assess the capacity of compound 3 (aslisted in Table 1) to activate Nurr1:RXR heterodimers. FIG. 1D shows theresults of a luciferase assay to assess the specificity of compound 3for Nurr1:RXRα heterodimers over other RXRα-containing heterodimers.FIG. 1E demonstrates that compound 3 selectively potentiates Nurr1:RXRαactivity over other heterodimers containing the RXRα subunit. Gal4 DNAbinding domain fusions with various RXRα and PPARg, VDR RXRα, RXRγ andNurr1 ligand binding domains activated with compound 3 show that onlyNurr1:RXRα heterodimers are activated by compound 3.

FIG. 1F demonstrates that compound 3 administration in mice activatescjun expression as assessed by qPCR. FIG. 1G shows that compound 3administration in mice activates tyrosine hydroxylase expression asassessed by qPCR.

FIGS. 2A-2G show the biochemical activity of compounds evaluated inneuroprotection assays. FIG. 2A demonstrates that compound 3 protectsNeuro2A cells from MPP+ induced toxicity. FIG. 2B shows that compound 3protects SHSY-5Y cells from low MPP+ (0.5 mM) induced toxicity. Thegraph in FIG. 2B shows, from left to right, control, XCT, and compound3. FIGS. 2C and 2D show that compound 3 protects SHSY-5Y cells from highMPP+(4 mM) induced toxicity. The graph in FIG. 2C shows, from left toright, control, XCT, and compound 3. FIG. 2E demonstrates that aretrovirus carrying shNurr1 is capable of infecting SHSY-5Y cells andlowering endogenous Nurr1 mRNA levels by ˜60% assessed by qPCR. FIG. 2Fshows that a retrovirus carrying shNurr1 is capable of infecting SHSY-5Ycells, lowering endogenous Nurr1 mRNA levels, and in turn attenuatingthe response of Nurr1:RXRα heterodimers to compound 3, indicatingactivity is based on Nurr1. The graph in FIG. 2F shows, from left toright, control, shNurr1, control compound 3, and shNurr1 compound 3.FIG. 2G shows that a retrovirus carrying shNurr1 infected SHSY-5Y cellslowers endogenous Nurr1 mRNA levels and lowers neuroprotective effect ofcompound 3, indicating activity is based on Nurr1. The graph in FIG. 2Gshows, from left to right, control, control+compound 3, shNurr1, andshNurr1+compound 3.

FIGS. 3A-3D show that C57/BL6 or TH promoter driven ASYN 120 transgenicmice injected intraperitoneally (IP) with 20 mg/kg compound 3 exhibit anincrease in dopamine (DA) and 5HT (serotonin) levels 4 h post injection.Vehicle IP injections were used as control. FIG. 3A shows that C57/BL6mice injected IP with 20 mg/kg compound 3 exhibit increased THexpression levels in comparison with vehicle IP injections as determinedby qPCR. FIG. 3B shows that C57/BL6 mice injected IP with 20 mg/kgcompound 3 exhibit increased striatum DA levels and DA metabolite(DOPAC, HVA) levels in comparison with vehicle IP injections asdetermined by HPLC. FIG. 3C shows that C57/BL6 mice injected IP with 20mg/kg compound 3 exhibit increased striatum 5HT levels and 5HTmetabolite 5HIAA levels in comparison with vehicle IP injections asdetermined by HPLC. FIG. 3D shows that C57/BL6 mice injected with 20mg/kg compound 3 do not exhibit increased striatum noradrenaline levelsin comparison with vehicle IP injections as determined by HPLC.

FIGS. 4A-4E show the effect of tested compounds on mouse behavior. FIG.4A shows C57/BL6 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-treated mice injected once IP with 20 mg/kg compound 3 7 daysafter MPTP treatment increase movement coordination as compared tovehicle IP injections as determined by rotarod experiments done on aColumbus Instruments apparatus. FIG. 4B shows C57/BL6 6OHDA-treated miceinjected once IP with 20 mg/kg compound 3 20 days after 6OHDA treatmentincrease movement coordination vs. vehicle IP injections as determinedby rotarod experiments done on a Columbus Instruments apparatus. FIG. 4Cshows C57/BL6 6OHDA-treated mice injected daily for 14 days IP with 20mg/kg compound 3 starting 20 days after 6OHDA treatment do not induceabnormal involuntary movements (AIMS) vs. daily L-DOPA IP injections.FIG. 4D shows C57/BL6 wt mice injected once IP with 20 mg/kg compound 3starting 20 days after CMS treatment increase swim times vs. vehicle IPinjections in the Porsolt forced swim test. FIG. 4E shows C57/BL6 wtmice injected once IP with 20 mg/kg compound 3 starting 20 days afterCMS treatment decrease floating times vs. vehicle IP injections in thePorsolt forced swim test.

FIGS. 5A-5E show the biochemical activity of compounds evaluated inneuroprotection experiments. FIGS. 5A and 5B show C57/BL6 6OHDA-treatedmice injected daily IP with 20 mg/kg compound 3 for 20 days after MPTPtreatment increase midbrain dopaminergic neuron numbers vs. C57/BL66OHDA-treated mice injected daily IP with vehicle as determined bycomputer-assisted image analysis system. The graph in FIG. 5A shows,from left to right, control, control compound 3, 6-OHDA, and 6-OHDAcompound 3. FIGS. 5C and 5D show C57/BL6 MPTP-treated mice injecteddaily IP with 20 mg/kg compound 3 for 7 days after MPTP treatmentincrease midbrain dopaminergic neuron numbers vs. C57/BL6 MPTP-treatedmice injected daily IP with vehicle as determined by computer-assistedimage analysis system. The graph in FIG. 5C shows, from left to right,control, control compound 3, MPTP, and MPTP compound 3. FIG. 5E showsNurr1 heterozygote 129/SV MPTP-treated mice injected daily IP with 20mg/kg compound 3 for 7 days after MPTP treatment do not exhibitincreased midbrain dopaminergic neuron numbers in comparison withC57/BL6 MPTP-treated mice injected daily IP with vehicle and incomparison with 120/SV control mice as determined by computer-assistedimage analysis system. The graph in FIG. 5E shows, from left to right,wt vehicle, wt MPTP, wt MPTP compound 3, Nurr1 het vehicle, Nurr1 hetMPTP, and Nurr1 het MPTP compound 3.

FIGS. 6-66 show ¹H and ¹³C NMR spectra for example compounds 1-46.Example numbers listed in these figures refer to the number assigned toeach compound in Table 1.

FIG. 67 shows the relative activation of Nurr1 and Nur77 by XCT0135908as assessed using a luciferase reporter assay.

FIGS. 68A and 68B show the pharmacokinetic profile of XCT in bloodplasma and in the brain of mice injected with this compound.Intraperitoneal (IP) or intracerebroventricular (ICV) XCT0135908 (1 and10 mg/kg) injections did not achieve any downstream effect in themidbrain, failing to upregulate the expression of c-jun or of tyrosinehydroxylase (TH). IP administration of XCT0135908 in mice (1 mg/kg)resulted in minimal penetration of the compound in the brain. Serialtail bleeds (n=3) were used for detection of the compound in plasma 1and 2 hours after IP administration. Similarly, mice (n=3) weresacrificed 1 and 2 hours after IP administration and brains weredissected. The concentrations of the parent compound in plasma and brainwere determined by LC-MS/MS.

FIGS. 69A-69C show the brain penetration, half-life, and c-juntranscriptional activation of compound 3. FIG. 69A shows the detectionof compound 3 in plasma 1, 2, 4, 8 and 24 hours after IP administration(n=5) to mice. FIG. 69B shows that IP administration of compound 3resulted in appreciable penetration of the compound in the brain. Mice(n=5) were sacrificed 1, 2, 4, 8 and 24 hours after IP administrationand brains were dissected 1, 2, 4, 8 and 24 hours post administration.The concentrations of the parent compound in plasma and brain weredetermined by LC/MSMS. FIG. 69C shows c-jun transcriptional activationin the midbrain as determined by qPCR 2 hours after IP administration ofcompound 3.

FIGS. 70A and 70B show the c-jun activating effects and neuroprotectiveproperties of compound 46. FIG. 70A shows that administration by IPinjection to mice revealed significant c-jun transcriptional activationin the midbrain as determined by qPCR 2 hours after administration. FIG.70B shows the results of experiments conducted to assess whethercompound 46 possesses neuroprotective properties. Compound 46 (12.5 μM)was added to human origin SHSY-5Y dopaminergic cells in which death wasinduced by the mitochondria complex I inhibitorMPP+(1-methyl-4-phenylpyridinium), the active metabolite of MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Pretreatment of thecells with compound 46 for 12 to 24 hours prior to incubation with MPP+significantly increased the survival of cells against varyingconcentrations of the toxic stimulus. Most of the MPP+-treated cellsreceiving vehicle died, while the few surviving cells remained attachedto the plate but were rounded and had lost all projections. Thesemorphological changes indicated impaired neuronal function. On thecontrary, cells treated with compound 46 appeared healthy, flattened,and well-attached, and their projections remained intact.

FIG. 71 shows a schematic representation of the intermolecularinteractions between compound 3 within the RXRα binding pocket (PDB ID:1MV9). Relevant hydrogen bonds and amino acids are indicated.

FIGS. 72A-72R show the ability of compound 3 to induce the expression ofgenes involved in dopamine biosynthesis as well as the neuroprotectiveeffects of compound 3 in cell lines and patient iPCs-deriveddopaminergic neurons. FIG. 72A shows coordinated expression levels ofthe three dopamine biosynthesis genes (TH, MDC and GCH1) mediated byactivation of Nurr1:RXRα by compound 3 (12.5 μM), as assessed by qPCR(n=8). FIG. 72B shows SHSY-5Y cell viability increases with compound 3treatment, as measured by MTT assay, after exposure to varyingconcentrations (0-16 μM) of hydrogen peroxide. FIG. 72C shows compound 3concentration-dependent SHSY-5Y cell viability, measured by MTT assay,after exposure to varying concentrations (0-4 mM) of MPP+. FIG. 72Dshows compound 3-mediated SHSY-5Y cell viability against MPP+(2 mM) isdependent on Nurr1 levels, as determined by retroviral knockdown ofNurr1. FIG. 72E shows that compound 3 treatment of primary rat corticalneurons reduces apoptotic death induced by co-transfecting a LRRK2G2019S or wild-type LRRK2 cDNA with CMV GFP, as measured by DAPIstaining. FIGS. 72F, 72G, 72H, and 72I show representative confocalmicroscopy images of the compound 3-treated primary rat corticalneurons. FIG. 72J shows the survival of human iPS cell-deriveddopaminergic neurons exposed to MPP+(0.5 and 1.0 mM) and receivingeither vehicle or compound 3 treatment. FIGS. 73K and 72L show thatcompound 3 treatment preserves dopaminergic neuron projections asdetermined by TH immunofluorescence of surviving iPS cell-derivedDAergic neurons after exposure to MPP+. FIG. 72M-72R show the effects ofcompound 3 on PD patient iPS cell-derived dopaminergic neurons with theLRRK2-G2019S mutation or corrected mutation (GC). Compound 3 treatmentrescues neurite length (M, expressed in nm), number (N) and branching(O) phenotypes. Representative images show staining with TuJ, TH andDAPI (FIGS. 72P, 72Q, and 72R).

FIGS. 73A-73F show the ability of compound 3 to protect dopaminergicneurons against MPTP toxicity. FIG. 73A shows the results of a Rotarodtest. Increased motor coordination was observed for C57BL/6 mice exposedto MPTP and receiving compound 3 treatment for 7 days.

FIG. 73 shows images of TH ICH in the substantia nigra of controlC57BL/6 mice or mice exposed to MPTP and receiving either vehicle orcompound 3 treatment. FIG. 73C shows stereological counts of substantianigra TH(+) neurons of control C57BL/6 mice or mice exposed to MPTP andreceiving either vehicle or compound 3 treatment. FIG. 73D shows imagesof TH ICH of substantia nigra dopaminergic projections to the striatumin C57BL/6 mice exposed to MPTP and receiving either vehicle or compound3 treatment. FIG. 73E shows the quantification of TH ICH dopaminergicprojections to the striatum of C57BL/6 mice exposed to MPTP andreceiving either vehicle or compound 3 treatment. FIG. 73F showsstereological counts of substantia nigra TH(+) neurons of controlwild-type 129SV mice or mice exposed to MPTP and receiving eithervehicle or compound 3 treatment (first three bars) and of Nurr1+/− 129SVmice exposed to MPTP and receiving either vehicle or compound 3treatment (last three bars).

FIGS. 74A-74L show the neuroprotective effects of compound 3 against6-OHDA and AAV-ASYN toxicity in mice. FIG. 74A shows a decreased numberof apomorphine-induced contralateral turns in C57BL/6 mice injectedunilaterally with 6-OHDA and treated daily with either vehicle orcompound 3 for 13 days, showing over 8-fold improvement (n=5). FIG. 74Bshows the results of a Rotarod test of mice subjected to unilateralinjection of 6-OHDA and receiving either vehicle or compound 3, showing12-fold improvement with compound 3 treatment (n=5). FIG. 74C shows SNTH ICH images of mice that received unilateral injection of 6-OHDA andwere treated with either vehicle or compound 3. FIG. 74D showsstereological counts of SN TH(+) neurons of mice receiving unilateralinjections of 6-OHDA and treated daily either vehicle or compound 3.Compound 3 treatment increased the number of TH(+) neurons by 47% (n=5).FIG. 74E shows striatum TH ICH of mice that received 6-OHDA injectionsand were treated with either vehicle or compound 3, showing 10-foldincreased innervation (FIG. 74F) (n=5). FIG. 74G shows SN TH ICH imagesof mice that received unilateral injections of AAV-ASYN andcontra-lateral injections of AAV-GFP and were treated with eithervehicle or compound 3. FIG. 74H shows stereological counts of SN TH(+)neurons of mice that received unilateral injections of AAV-ASYN and weretreated daily either with vehicle or compound 3. Compound 3 increasedthe number of TH(+) neurons by 48% (n=7). FIG. 74I shows the striatum THICH of mice that received injections of AAV-ASYN or AAV-GFP and weretreated with either vehicle or compound 3, showing 10-fold increasedinnervation (FIG. 74J) (n=7).

FIGS. 75A-75J show that compound 3 induces dopamine biosynthesis andsymptomatic relief without dyskinesias in vivo in PD mouse models. FIG.75A shows the results of qPCR assays conducted to assess TH expressionlevels in mouse midbrain 4 hours after vehicle or compound 3 (10 mg/kg)IP administration. FIG. 75B shows dopamine and dopamine metabolitelevels 4 hours after IP administration of vehicle or compound 3 (10mg/kg) in wild-type mice as assessed by HPLC. FIG. 75C shows dopamineand dopamine metabolite levels 4 hours after IP administration ofvehicle or compound 3 (10 mg/kg) in alpha synuclein transgenic mice asassessed by HPLC (n=4). FIG. 75D shows noradrenaline levels 4 hoursafter IP administration of vehicle or compound 3 (10 mg/kg) as assessedby HPLC. FIGS. 75E and 75F show schematic representations of thecompound 3 treatment regimens. FIG. 75G shows the accelerating rotarodlatency times of MPTP-treated mice (4-40 rpm over 5 min) 4 hours afterIP administration of vehicle or compound 3 (10 mg/kg). FIG. 75H showsthe accelerating rotarod latency times of 6-OHD-treated mice (4-40 rpmover 5 min) 4 hours after IP administration of vehicle or compound 3 (10mg/kg). FIG. 75I shows spontaneous contralateral turns per minute of6-OHDA-treated mice 4 hours after IP administration of vehicle, compound3 (10 mg/kg) or L-DOPA. FIG. 75J shows a schematic representation of thecompound 3/L-DOPA treatment regimen.

DETAILED DESCRIPTION

The present invention provides substituted pyrimidines that are, forexample, effective as activators of the Nurr1:RXRα heterodimer.Substituted pyrimidines of formula (I) are selective agonists of theNurr1:RXRα heterodimer and can inhibit neuronal degeneration andsymptoms usually observed in Parkinson's disease:

wherein n is 0 to 2, p is 0 to 2, X is N(R₆), or 0;Cy is a phenyl ring or a heteroaromatic 5- or 6-membered ring containingbetween 1 and 4 heteroatoms (e.g., selected from O, N, and 5);each R₁ is independently selected from the group consisting of halogen,cyanate, isocyanate, thiocyanate, isothiocyanate, selenocyanate,isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano, nitro,hydroxy, acyl, mercapto, carboxyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR₇,—SR₇, and —N(R₈)R₇;each R₂ is independently selected from the group consisting of halogen,cyanate, isocyanate, thiocyanate, isothiocyanate, selenocyanate,isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano, nitro,hydroxy, acyl, mercapto, carboxyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR₇,—SR₇, and —N(R₈)R₇;R₃ is selected from the group consisting of hydrogen, halogen, cyanate,isocyanate, thiocyanate, isothiocyanate, selenocyanate,isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano, nitro,hydroxy, acyl, mercapto, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, OR₇, —SR₇,—N(R₈)R₇, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted cycloalkyl and optionally substitutedheterocyclyl, or R₃ and R₆, when present, combine with the atoms towhich they are bound to form an optionally substituted 5-memberedheteroaryl or heterocyclyl;R₄ is selected from the group consisting of hydrogen, halogen,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted aryl-alkyl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted heteroaryl-alkyl, optionally substitutedheterocyclylalkyl, OR₇, and —N(R₈)R₇;R₅ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, and optionallysubstituted aryl;R₆ is selected from the group consisting of hydrogen and optionallysubstituted alkyl;each R₇ is independently selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted aryl, optionally substituted aryl-alkyl, and optionallysubstituted heterocyclyl; andeach R₈ is independently selected from the group consisting of hydrogenand optionally substituted alkyl.

The compounds of formula (I), wherein R₅ is H, are carboxylic acids thatcan be used in the form of free acids or in the form of salts, which canresult from the combination of the acid with an inorganic base, orpreferably with a pharmaceutically acceptable, non-toxic organic base.Inorganic bases that can be used to produce an addition salt of theinvention include, without limitation, the hydroxides of sodium,potassium, magnesium, and calcium. Organic bases that can be used toproduce an addition salt of the invention include, without limitation,amines, amino alcohols, basic amino acids such as lysine or arginine, orcompounds bearing a tertiary amine function, such as betaine or choline.Salts of formula (I) with an inorganic or organic base can be obtainedin conventional manner using well known methods to those skilled in theart, e.g., by mixing stoichiometric amounts of an acid of formula (I)(wherein R₅ is H) and a base in a solvent such as water or ahydroalcoholic mixture and then lyophilizing the resulting solution.

Esterified compounds of formula (I) that can be used in the form of aprodrug are pharmaceutically acceptable esters that can be produced bythe derivatization of the carboxylic acid with an appropriatelysubstituted aliphatic or aromatic alcohol or an alkyl halide.

Preferred embodiments of substituted pyrimidines include, for example,compounds in which R₃ is not hydrogen, such as compounds in which Cy isa phenyl ring, R₃ is optionally substituted alkyl or optionallysubstituted alkenyl, R₄ is optionally substituted methyl (e.g.,halomethyl), and R₅ is hydrogen or optionally substituted alkyl. Forinstance, in some preferred embodiments, Cy is a phenyl ring, R₃ isalkyl or alkenyl, R₄ is methyl or trifluoromethyl, and R₅ is hydrogen oralkyl.

Preferred embodiments of substituted pyrimidines also include compoundsof formula (1a):

wherein each of n, p, and R₁-R₆ are as defined above.

Preferred embodiments of substituted pyrimidines additionally includecompounds of formula (1b):

or a free base thereof,wherein each of n, p, and R₁-R₄ and R₈ are as defined above; and L isselected from the group consisting of —O—, —O—R₉—O—, and —O—R₉—, whereinR₉ is an optionally substituted alkyl, optionally substituted alkenyl,or optionally substituted alkynyl moiety.

Preferred embodiments of substituted pyrimidines further includecompounds of formula (1a) in which R₄ is haloalkyl, e.g., CF₃. Forexample, preferred embodiments of substituted pyrimidines includecompounds of formula (1c):

wherein each of n, p, and R₁-R₃, R₅, and R₆ are as defined above.

Preferred embodiments of substituted pyrimidines also include compoundsof formula (1d):

wherein each of n, p, and R₁-R₅ are as defined above.

Preferred embodiments of substituted pyrimidines also include compoundsof formula (1e):

wherein each of n, p, R₁, R₂, R₄, and R₅ are as defined above;R₁₀ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedaryl-alkyl, optionally substituted heteroaryl, optionally substitutedcycloalkyl, and optionally substituted heterocyclyl; andR₁₁ is selected from the group consisting of optionally substitutedalkyl, optionally substituted aryl, optionally substituted aryl-alkyl,optionally substituted heteroaryl, optionally substituted cycloalkyl,and optionally substituted heterocyclyl.

Preferred embodiments of substituted pyrimidines further includecompounds of formula (1f):

or a salt thereof, where each of n, p, and R₁-R₄ and R₆ are as definedabove; and Y is —O—R₉—, wherein R₉ is an optionally substitutedalkylene, optionally substituted alkenylene, or optionally substitutedalkynylene chain.

For instance, preferred embodiments of substituted pyrimidines includecompounds of formula (1g):

or a salt thereof, where each of n, p, and R₁-R₄ and R₆ are as definedabove.

Additional preferred embodiments of substituted pyrimidines includecompounds of formula (1h):

or a salt thereof, where each of n, p, and R₁-R₄ andR₆ are as defined above.

Methods of Treatment Neurodegenerative Disorders and Nurr1

Parkinson's disease is a common neurodegenerative disease, and itspathology is characterized by the progressive loss of the dopaminergic(DAergic) neurons of the substantia nigra (SN), leading to striataldopamine (DA) deficiency (Meissner, Rev. Neurol. 168:809-814, 2012).

Current medications replenish dopamine, and offer temporary symptomaticrelief to patients. However, chronic treatments cause serious sideeffects in almost all patients, including abnormal involuntary movements(AIMs) and dyskinesias, limiting their efficacy (Athauda et al. NatureReviews Neurology 11:25-40, 2015). Moreover, DA replacement does notimpede neurodegeneration, and PD pathology progresses (Fahn. J. NeuralTransm. Suppl. 70:419-426, 2006). Despite considerable progress in ourunderstanding of PD pathophysiology, pharmacological strategies toprevent neurodegeneration remain elusive, and the disease remainsincurable. Therefore, validation of novel targets that diminish DAreplacement side-effects and halt or slow disease progression is anurgent unmet clinical need (Meissner et al. Nat. Rev. Drug Discov.10:377-393, 2011). Nurr1 (NR4A2), a nuclear receptor, has been apromising target candidate for therapy, as it is implicated in both DAbiosynthesis and DAergic neuron survival. Nurr1 is expressed indeveloping and mature dopaminergic neurons, and it is required for bothsurvival and final complete dopaminergic differentiation (Zetterström etal. Science 276:248-250, 1997 and Saucedo-Cardenas et al. Proc. Natl.Acad. Sci. USA. 95:4013-4018, 1998). Nurr1 enhances in vitro and in vivotranscription of tyrosine hydroxylase (TH), the rate-limiting enzyme ofDA biosynthesis, and of GTP cyclohydrolase I (GCH1), the first enzyme inthe biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor forTH activity (Kim et al. J. Neurochem. 85:622-634, 2003 and Gil et al. JNeurochem 101:142-150, 2007). Decreased Nurr1 levels have been stronglyassociated with PD and reduced DAergic neuron survival. Nurr1 ablationin adult mice leads to dystrophic dopaminergic axons (Kadkhodaei et al.J. Neurosci. 29:5923-5932, 2009), loss of striatal DA (Kadkhodaei et al.Proc. Natl. Acad. Sci. USA. 110:2360-2365, 2013) and behavioral featuresof parkinsonism during aging (Zhang et al. Neurobiol. Aging 33:7-16,2012). Nurr1 mutations decreasing its mRNA have been found in familialand sporadic PD patients (Le et al. Nat. Genet. 33:85-89, 2003 andSleiman et al. Neurosci. Lett. 457:75-79, 2009). Given the role of Nurr1in PD, we demonstrate that selective activation of Nurr1 by compounds ofthe invention represents a monotherapeutic treatment paradigm for PDthat offers both disease modification and symptomatic relief.

Nurr1 binds to DNA as a monomer, homodimer or heterodimer with RXRα orRXRγ. Since in midbrain dopaminergic neurons Nurr1 heterodimerizes withRXRα (Wallen-Mackenzie et al. Genes Dev. 17:3036-3047, 2003), syntheticligands that bind to the RXRα binding pocket are the preferred approachto neurodegenerative disease therapy described herein. RXRα is aheterodimer partner of several nuclear receptors, and existing syntheticRXRα ligands activate several RXRα heterodimers (Pérez et al. Biochim.Biophys. Acta 1821:57-69, 2012). Two such ligands, XCT0135908 andbexarotene (Wallen-Mackenzie et al. Genes Dev. 17:3036-3047, 2003 andCramer et al. Science 335:1503-1506, 2012) have been shown to activateNurr1:RXRα but also other RXRα heterodimers. Bexarotene, an approvedantineoplastic activating RXRα heterodimers with LXR, PPARγ and Nurr1,had promising results in animal models of Alzheimer's disease and PD(Cramer et al. Science 335:1503-1506, 2012 and McFarland et al. ACSChem. Neurosci. 4:1430-1438, 2013) however, failed to be replicated(Landreth et al. Science 340:924-g, 2013 and Volakakis et al. J.Neurosci. 35:14370-14385, 2015). In vitro, XCT0135908 (Wallen-Mackenzieet al. Genes Dev. 17:3036-3047, 2003), activates Nurr1:RXRα heterodimersand RXRα:RXRα homodimers (McFarland et al. ACS Chem. Neurosci.4:1430-1438, 2013), but its bioavailability has not been reported.

As described herein, compounds of the invention meet an important unmetclinical need, as compounds of the invention are highly bioavailable,blood-brain barrier penetrant, and selectively potentiate the activityof the Nurr1:RXRα heterodimer, thereby modulating dopamine biosynthesisand other important gene expression profiles while minimizingpotentially undesirable off-target effects.

Gene Modulating Effects and Neuroprotective Effects of Compounds of theInvention

Compounds of the invention can be used to treat or inhibit theprogression of a disease or condition that involves the activity of thetranscription co-activator Nurr1. Nurr1 forms a heterodimeric proteincomplex with RXRα, and this ensemble modulates the expression of avariety of genes that collectively regulate dopaminergic neuron activityand differentiation in the brain. Attenuated activity of the Nurr1transcription co-activator has been correlated with the onset ofParkinson's disease as well as symptoms associated with Parkinson'sdisease that are not limited to patients presenting with this ailment,such as bradykinesia, dyskinesia, learning difficulties, memorydisorders, attention deficit disorder, insomnia, and katalepsy.Compounds of the present invention can thus be used to treat any or allof these symptoms, either in a patient suffering from Parkinson'sdisease or in a patient presenting with a different neurodegenerativedisorder. Compounds of the invention can also be used to treat orinhibit the progression of a disease characterized by aberrantbiosynthesis of one or more products of Nurr1 signalling, such asL-dopamine, as well as diseases and conditions in which the activity ofNurr1 is impaired or aberrant relative to a healthy subject, such asthose characterized by a reduction in the quantity and activity ofdopaminergic neurons. By promoting L-dopamine biosynthesis and release,compounds of the invention are capable of halting neurodegeneration ofsignificant neurons, such as dopamine midbrain neurons and other neuronsof the dopaminergic system. For instance, a lack of dopamine and loss ofdopaminergic neuron projections to the striatum is manifested inactivity alterations in the basal ganglia that regulate movement.Moreover, dopamine depletion from non-striatal dopamine pathways mayunderlie neuropsychiatric pathology associated with Parkinson's disease.Compounds of the invention can be administered to a patient in order toinduce neuroprotection in any or all of these areas.

Due to their Nurr1-stimulating activity, compounds of the invention arecapable of increasing the rate of transcription of polynucleotides(e.g., genes) encoding enzymes involved in dopamine biosynthesis, suchas tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC),and phenylalanine hydroxylase (PheOH, alternatively PheH or PAH).Compounds of the instant invention are also capable of enhancing therate of transcription of polynucleotides (e.g., genes) encoding enzymesinvolved in tetrahydrobiopterin (BH4) biosynthesis, such as GTPcyclohydrolase (GCH1). Compounds of the invention are also capable ofincreasing the rate of translation of the RNA resulting from theabove-described transcription processes and thus increasing the amountof functional protein produced.

The compounds of the invention are unique in that they can selectivelypotentiate the activity of the Nurr1:RXRα heterodimer over a series ofrelated transcription co-activator complexes that include RXRα. Knownbinding partners of RXRα include Nurr1, vitamin D receptor (VDR),peroxisome proliferator-activated receptor γ (PPARγ), RXRα, and RXRγ.The compounds of the invention thus represent an improvement overpreviously reported therapeutics, as the compounds of the presentinvention are capable of discriminating among these transcriptionco-activator complexes and selectively modulating Nurr1:RXRα activity.Additionally, compounds of the present invention are uniquely suitablefor administration to a subject in vivo, as these compounds are readilybioavailable and are capable of inducing Nurr1-dependent neuroprotectionin subjects.

Besides the dopaminergic system, the serotoninergic (5-HT) system andraphe nucleus (Braak et al., 2003) is also thought to be involved in thenon motor PD symptoms. In the normal brain, there is a denseserotonergic innervation of the basal ganglia from the raphe nuclei.Indeed, in early postmortem studies of patients with PD, there isdepletion of serotonin in the caudate as well. Imaging studies in vivohave also suggested depletion of 5-HT innervation to the striatum asmeasured via decreased serotonin transporter binding. The loss ofstriatal 5-HT in PD may be secondary to neurodegeneration as Lewy bodiesare seen in the raphe nuclei and there is associated cell loss.

Surprisingly, compounds of the invention are also capable ofpotentiating the biosynthesis and release of serotonin by exertingactivating effects on the serotoninergic (5-HT) system and raphe nucleusof the brain. This region is thought to be involved in the propagationof non-motor Parkinson's disease symptoms. Compounds of the inventioncan thus be used to treat symptoms mediated by the release and activityof serotonin, such as anxiety and depression that are often observed inpatients suffering from Parkinson's disease. Significantly, thecompounds of the present invention represent the first reported examplesof therapeutics that are capable of treating symptoms that occur due toNurr1 and 5-HT-mediated signaling.

Due to their serotoninergic system-stimulating activity, compounds ofthe invention are capable of increasing the rate of transcription ofpolynucleotides (e.g., genes) encoding enzymes involved in serotoninbiosynthesis, such as tryptophan hydroxylase (TPH) and amino aciddecarboxylase. Compounds of the invention are also capable of increasingthe rate of translation of the RNA resulting from these transcriptionprocesses and thus increasing the amount of functional protein produced.

The current paradigm for treating Parkinson's disease primarily focuseson treating the symptoms associated with Parkinson's disease. To date,drug discovery efforts for PD have traditionally aimed either onsymptomatic DA replacement treatments and alleviating dyskinesias or onneuroprotection. Existing DA replacement therapies may improve PDsymptoms; however, they do not offer disease modification, and chronicuse causes severe side effects such as dyskinesias (Rascol et al. Mov.Disord. 30:1451-1460, 2015 and Schapira. CNS Drugs 25:1061-1071, 2011).For instance, L-DOPA, has traditionally been administered to patientswith Parkinson's disease in order to promote an increase in dopaminebiosynthesis and release. Neuroprotective approaches, while they aim atthe core of PD pathology, so far have not delivered the expected results(Stocchi. Neurotherapeutics 11:24-33, 2014). The compounds of theinstant invention are unique in that they not only provide a symptomatictreatment modality but can also be administered to a patient withParkinson's disease in order to induce a sustained neuroprotectiveeffect in the dopaminergic and serotoninergic systems of the brain.These compounds can therefore be administered to a patient, e.g., at theonset of one or more symptom of the disease, such as bradykinesia,dyskinesia, insomnia, or katalepsy, among others, in order to alleviatethese symptoms. Additionally or alternatively, compounds of theinvention may be administered to a patient over the course of aprolonged treatment regimen in order to provide long-term relief fromthe disease by inducing neuroprotection in dopaminergic andserotoninergic systems of the brain.

Target Specificity

Compounds of the invention surprisingly exhibit an important biologicalbenefit, as these compounds are capable of selectively activating theNurr1:RXRα heterodimer over a series of closely related RXRα-containingtranscription factors. In this way, compounds of the invention maymodulate dopamine and serotonin biosynthesis and induce neuroprotectionwithout promoting potentially undesirable off-target effects. Compoundsof the invention therefore satisfy an unmet clinical need, as previousNurr1 agonists lack this target selectivity. For example, XCT0135908(also referred to herein as “XCT”) is a known Nurr1 activator describedin, e.g., WO 2005/047268. This compound has the structure shown below:

XCT activates Nurr1:RXRα heterodimers as well as RXRα:RXRα homodimers(McFarland et al. ACS Chem. Neurosci. 4:1430-1438, 2013). Additionally,present data show that XCT preferentially activates Nur77:RXRαheterodimers and not Nurr1:RXRα heterodimers. Thus, XCT does notselectively activate the Nurr1:RXRα heterodimer (FIG. 67). In contrast,compounds described herein, such as compound 3, selectively potentiatethe activity of the Nurr1:RXRα homodimer.

As described herein, compounds of the invention, such as compound 3, arecapable of specifically activating the Nurr1:RXRα complex over closelyrelated potential targets, including other RXRα-containing heterodimers.As reported below, the ligand binding domains of Nurr1, RXRα and avariety of other nuclear receptors were cloned to create GAL4 chimeras,and the ligand binding domain of RXRα was fused with VP16. Thesemolecular chimeras were co-transfected in pairs with the RXRα:VP16 alongwith a GAL4-responsive luciferase reporter into SHSY-5Y dopaminergiccells and were assayed for their ability to be activated by compounds ofthe invention. Compounds described herein readily activate Nurr1GAL4:RXRαVP16 heterodimer chimeras without activating RXRαGAL4 homodimerchimeras, indicating again that these compounds are Nurr1:RXRα-selectiveagonists. For example, RXRαVP16 heterodimer chimeras with VDRGAL4,RXRγGAL4 or PPARγGAL4 were not activated by compound 3, as this compoundwas capable of selectively activating the Nurr1:RXRα heterodimer. Thesedata indicate the high degree of selectivity of compounds of theinvention for Nurr1GAL4:RXRαVP16 heterodimers and the lack of activitytowards other closely related molecular targets. The selectivity ofcompounds of the invention for the Nurr1:RXRα heterodimer is attributedto the fact that the ligand binding pocket of RXRα varies slightlydepending on the heterodimer partner (Lionta et al. Curr. Top. Med.Chem. 14:1923-1938, 2014). Compounds of the invention are thereforecapable of discriminating among transcriptional activators andselectively potentiating the activity of the Nurr1:RXRα complex (see,e.g., FIG. 1E).

Bioavailability

In addition to selectively activating the Nurr1:RXRα heterodimer,compounds of the invention exhibit high bioavailability and are readilybrain-penetrant. The beneficial absorption and distribution profiles ofthe compounds of the invention represent an innovative solution to anunmet clinical problem, as previous Nurr1 agonists, such as XCT, lackthe ability to persist in vivo and to cross the blood-brain barrier. Forinstance, as reported in the examples below, XCT was administered tomice to test its bioactivity in vivo. Intraperitoneal (IP) orintracerebroventricular XCT (1 and 10 mg/kg) injections did not resultin any expression alterations of midbrain genes such c-jun or tyrosinehydroxylase at different time points after administration. LC-MS/MSanalysis of blood plasma or brain homogenates and targeted searching ofthe parent compound at 1 and 2 hours after IP XCT (1 μg/kg)administration indicated low compound stability and minimal brainpenetration (brain/blood<0.03; FIGS. 68A-B).

In contrast to XCT, compounds of the invention exhibit a highbioavailability and are readily blood-brain barrier penetrant. Forexample, IP administration of compound 3 (1 mg/kg) in mice revealed thatthis compound reaches the brain and exhibits an approximate half-life ofabout 2 hours in both blood and brain as assessed by LC-MS/MS(brain/blood concentration AUC ratio 1.7, FIGS. 69A-C). Additionally,compound 3 was biologically active in the brain, as administration ofthis compound resulted in increased midbrain c-jun expression (FIGS. 1Fand 69C). Similarly, compound 46 is highly bioavailable and capable ofcrossing the blood brain barrier. Administration by IP injection to micerevealed significant c-jun transcriptional activation in the midbrain asdetermined by qPCR 2 hours after administration (FIG. 70A). Thiscompound additionally exhibits a strong neuroprotective effect (FIG.70B).

Since specific Nurr1:RXRα activators have not been reported previously(Vaz B et al. Expert Opin. Drug Discov. 7:1003-1016, 2012), compounds ofthe invention represent a first-in-class approach to targetedneurodegenerative disease therapy. The compounds described herein arespecific for Nurr1:RXRα heterodimers, stable in vivo, andbrain-penetrant. These compounds exhibit combinatorial neuroprotectiveand symptomatic benefits in preclinical animal models, validatingselective Nurr1:RXRα heterodimer activation as a therapeutic paradigmfor the treatment of neurodegenerative disorders, such as Parkinson'sdisease.

Nurr1:RXRα is a Validated Target for Neurodegenerative Disease Therapy

Our experiments demonstrate that activation of Nurr1:RXRα by compoundsof the invention, such as compound 3 and compound 46, provideneuroprotection that can halt neuronal loss associated withneurodegenerative disorders, such as Parkinson's disease, in bothtoxin-based and genetic preclinical mouse models of Parkinson's disease.In addition, we demonstrate that the effects of compounds of theinvention, such as compound 3, both in vitro and in vivo depend on Nurr1expression. Compounds of the invention, such as compound 3, alsoincrease DA levels in vivo and offer symptomatic relief in twopost-degeneration PD animal models at the same dose. This dual activityindicates that, unlike most efforts for developing therapeuticapproaches for the treatment of Parkinson's disease, Nurr1:RXRαactivation offers a unique combined efficacy distinct from that of othertargets. Because of the divergent toxin and genetic insults we have usedin our PD models, our data indicate that various pathways leading to thedemise of dopaminergic neurons can be overcome using compounds of theinvention, such as compound 3 and compound 46. This implies coordinatedcontrol of a complex neuroprotective network by Nurr1:RXRα. Moreover, asopposed to the dopamine-excessive bursts induced by dopamine replacementtherapies, compounds of the invention, such as compound 3, finelyregulate dopamine production in a more physiological manner via thetranscriptional activation of the dopamine biosynthesis genes (TH, GCH1and MDC), without affecting dopamine catabolism and without elicitingdyskinesias.

Therapeutic uses of the compounds of the invention are unique in thatthey represent the first instances in which a single compound thatactivates a specific target achieves a dual therapeutic advantage fortreating neurodegenerative disorders, such as Parkinson's disease. Sincethe neuroprotective effects of compounds of the invention, such ascompound 3, extend to induced pluripotent stem cell (iPSc)-deriveddopaminergic neurons of Parkinson's disease patients, selectiveNurr1:RXRα activation provides a clear therapeutic benefit to patientssuffering from this condition.

Pharmaceutical Compositions

The compounds of the invention can be formulated into pharmaceuticalcompositions for administration to human subjects in a biologicallycompatible form suitable for administration in vivo. Accordingly, inanother aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of the invention in admixture with asuitable diluent, carrier, or excipient.

The compounds of the invention may be used in the form of the free base,in the form of salts or as prodrugs. All forms are within the scope ofthe invention. In accordance with the methods of the invention, thedescribed compounds or salts, or prodrugs thereof may be administered toa patient in a variety of forms depending on the selected route ofadministration, as will be understood by those skilled in the art. Thecompounds of the invention may be administered, for example, by oral,parenteral, buccal, sublingual, nasal, rectal, patch, pump, ortransdermal administration and the pharmaceutical compositionsformulated accordingly. Parenteral administration includes intravenous,intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal,intrapulmonary, intrathecal, rectal, and topical modes ofadministration. Parenteral administration may be by continuous infusionover a selected period of time.

A compound of the invention may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or it maybe enclosed in hard or soft shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, a compound of theinvention may be incorporated with an excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

A compound of the invention may also be administered parenterally.Solutions of a compound of the invention can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO and mixtures thereof with or without alcohol, and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington: The Science and Practice of Pharmacy (2012, 22^(nd) ed.) andin The United States Pharmacopeia: The National Formulary (2015, USP 38NF 33).

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that may be easily administered via syringe.

Compositions for nasal administration may conveniently be formulated asaerosols, drops, gels, and powders. Aerosol formulations typicallyinclude a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device, such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant, which can be a compressed gas,such as compressed air or an organic propellant, such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, where the active ingredient isformulated with a carrier, such as sugar, acacia, tragacanth, gelatin,and glycerine. Compositions for rectal administration are convenientlyin the form of suppositories containing a conventional suppository base,such as cocoa butter.

The compounds of the invention may be administered to an animal, e.g., ahuman, alone or in combination with pharmaceutically acceptablecarriers, as noted herein, the proportion of which is determined by thesolubility and chemical nature of the compound, chosen route ofadministration, and standard pharmaceutical practice.

Dosages

The dosage of the compounds of the invention, and/or compositionscontaining a compound of the invention, can vary depending on manyfactors, such as the pharmacodynamic properties of the compound; themode of administration; the age, health, and weight of the recipient;the nature and extent of the symptoms; the frequency of the treatment,and the type of concurrent treatment, if any; and the clearance rate ofthe compound in the animal to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors. Thecompounds of the invention may be administered initially in a suitabledosage that may be adjusted as required, depending on the clinicalresponse. In general, satisfactory results may be obtained when thecompounds of the invention are administered to a human at a daily dosageof, for example, between 0.05 mg and 3000 mg (measured as the solidform). Dose ranges include, for example, between 10-1000 mg (e.g.,50-800 mg). In some embodiments, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg of thecompound is administered. Preferred dose ranges include, for example,between 0.05-15 mg/kg or between 0.5-15 mg/kg.

Alternatively, the dosage amount can be calculated using the body weightof the patient. For example, the effective dose of a compound of theinvention can range, e.g., from about 0.0001 to about 100 mg/kg of bodyweight per single (e.g., bolus) administration, multiple administrationsor continuous administration. Alternatively, a compound of the inventionmay be administered in an appropriate dose to achieve a suitable serumconcentration, e.g., a serum concentration of 0.0001-5000 μg/mL persingle (e.g., bolus) administration, multiple administrations orcontinuous administration, or any effective range or value thereindepending on the condition being treated, the route of administrationand the age, weight, and condition of the subject. The dose may beadministered one or more times (e.g., 2-10 times) per day, week, month,or year to a patient (e.g., a human) in need thereof.

Compositions for Combination Therapy

A compound of the invention can be used alone or in combination withother agents that can be used to treat neurodegenerative disorders, suchas Parkinson's disease. A compound of the invention can be admixed withan additional active agent and administered to a patient in a singlecomposition (e.g., a tablet or capsule), or a compound of the inventioncan be administered to a patient separately from an additional activeagent. A compound of the invention and an additional active agent can besequentially administered to a patient as part of a dosing regimendescribed herein. For instance, a compound of the invention may beadmixed or formulated for co-administration with Levodopa(L-dihydroxyphenylalanine), L-aromatic amino acid decarboxylase (MDC)inhibitors, and/or catechol O-methyltransferase (COMT) inhibitors.Exemplary AADC inhibitors that may be included in a pharmaceuticalcomposition of the invention include Carbidopa((2S)-3-(3,4-dihydroxyphenyl)-2-hydrazinyl-2-methylpropanoic acid) andBenserazide(2-amino-3-hydroxy-N′-[(2,3,4-trihydroxyphenyl)methyl]propanehydrazide).Exemplary COMT inhibitors that may be included in a pharmaceuticalcomposition of the invention include Entacapone((E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethylprop-2-enamide),Tolcapone ((3,4-dihydroxy-5-nitrophenyl)-(4-methylphenyl)methanone), andNitecapone(3-[(3,4-dihydroxy-5-nitrophenyl)methylidene]pentane-2,4-dione).

In combination treatments, the dosages of one or more of the therapeuticcompounds may be reduced from standard dosages when administered alone.For example, doses may be determined empirically from drug combinationsand permutations or may be deduced by isobolographic analysis (e.g.,Black et al., Neurology 65:S3-S6, 2005). In this case, dosages of thecompounds when combined may provide a therapeutic effect.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsclaimed herein are performed, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention.

I. General Synthetic Methods

The compounds of formula (I) in which X is N(R₆), R₆═H, and R₅=alkyl, Hmay be prepared as described in Scheme 1. For example, alkylation ofβ-ketoester of formula (II) with an alkyl halide under standardconditions (i.e., NaOEt/EtOH) will result in an alkylated β-ketoester offormula (III). A compound of formula (III) can undergo a cyclizationreaction with an amidine of formula (IV) to afford a pyrimidinol offormula (V) (J. Org. Chem. 2007, 72, 5835-5838). Treatment of compoundof formula (V) with neat POCl₃ will generate a chloropyrimidine offormula (VI) (Chem. Pharm. Bull. 1982, 30, 4314-4324), which, uponcoupling with aminobenzoate of formula (VII) under acidic conditions(Bioorg. Med. Chem. 2006, 14, 7761-7773), will generate a compound offormula (I) in which R₆═H, R₅=alkyl. Ester hydrolysis under standardbasic conditions will produce an acid compound of formula (I) in whichR₅, R₆═H.

In another embodiment, the compounds of formula (I) in which X is N(R₆),R₆=alkyl, and R₅=alkyl, H may be prepared according to Scheme 1. Forexample, alkylation of the secondary nitrogen of compound of formula (I)in which R₆═H and R₅=alkyl with an alkyl halide (R—X) in the presence ofa base such as Cs₂CO₃ will generate an N-alkylated compound of formula(I) in which R₆=alkyl and R₅=alkyl, which can undergo an esterhydrolysis under standard basic conditions to afford an acid compound offormula (I) in which R₆=alkyl and R₅═H.

Preparation of a compound of formula (III): A compound of formula (II)was added to a solution of NaOEt in EtOH at room temperature understirring for 15 min. Then addition of an alkyl halide as a solution inEtOH followed, and the reaction mixture was refluxed for 4-5 h. When TLCindicated the consumption of compound of formula (II), the reactionmixture was concentrated under reduced pressure, and the resultingresidue was diluted with water and extracted with CH₂Cl₂. The combinedorganic layer was washed with water, dried over MgSO₄, filtered, andconcentrated under reduced pressure to give the crude product.Purification by flash chromatography on silica gel with Hex:EtOAc orvacuum distillation afforded the compound of formula (III).

Preparation of a compound of formula (V): A ketoester of formula (III)was added to a solution of NaOEt (21% w/w solution in EtOH) and anamidine hydrochloride of formula (IV) in EtOH. The reaction mixture wasstirred overnight under reflux and concentrated under reduced pressure.The resulting residue was treated with 1 N HCl and then extracted withCH₂Cl₂. The combined organic layer was washed with 1 N HCl, brine, driedover MgSO₄, filtered, and concentrated under reduced pressure to givethe crude product. Purification by crystallization or flash columnchromatography on silica gel with Hex:EtOAc afforded the compound offormula (V).

Preparation of a compound of formula (VI): A compound of formula (V) wasdissolved in neat POCl₃, and the resulting solution was stirred for 6 hunder reflux. The solvent was evaporated under reduced pressure, and theresulting residue was dissolved in EtOAc and then washed with saturatedNa₂CO₃ and brine. The organic layer was dried over MgSO₄, filtered, andconcentrated under reduced pressure to give the crude product.Purification by flash chromatography on silica gel with Hex:EtOAcafforded the compound of formula (VI).

Preparation of a compound of formula (I) in which R₆═H, R₅=alkyl: Asolution of a compound of formula (VI), an aminobenzoate of formula(VI), and a catalytic amount of concentrated HCl in EtOH was stirredunder reflux. The reaction progress was monitored by TLC, and catalyticamounts of concentrated HCl were gradually added to the reaction mixturein order to drive the reaction to completion. When TLC indicated theconsumption of compound of formula (VI), the solvent was evaporatedunder reduced pressure. The resulting residue was treated with waterfollowed by extraction with EtOAc. The combined organic layer was washedwith water, dried over MgSO₄, filtered, and concentrated under reducedpressure to give the crude product. Purification by flash chromatographyon silica gel with Hex:EtOAc afforded the compound of formula (I) inwhich R₅=alkyl, R₆═H.

Preparation of compound of formula (I) in which R₅, R₆═H: A solution ofcompound of formula (I) in which R₅=alkyl, R₆═H in EtOH was treated with1 N LiOH and then stirred at room temperature overnight. The solvent wasevaporated under reduced pressure, and the resulting residue was treatedwith 1 N NaOH and then extracted with Et₂O. The remaining aqueous layerwas acidified with 1 N HCl to pH˜2-3 and then extracted with EtOAc. Thecombined organic layer was washed with 0.1 N HCl, dried over MgSO₄,filtered, and concentrated under reduced pressure to afford carboxylicacid compound of formula (I) in which R₅, R₆═H.

Preparation of a compound of formula (I) in which R₅, R₆=alkyl: Asolution of a compound of formula (I) in which R₆═H, R₅=alkyl in DMF wastreated with Cs₂CO₃, and then an alkyl halide was added. The reactionmixture was stirred at room temperature overnight and then diluted withwater followed by extraction with EtOAc. The combined organic layer waswashed with water, dried over MgSO₄, filtered, and concentrated underreduced pressure to give the crude product. Purification by flashchromatography on silica gel with Hex:EtOAc afforded the ester compoundof formula (I) in which R₅, R₆=alkyl.

Preparation of a compound of formula (I) in which R₆=alkyl, R₅═H: Asolution of an ester compound of formula (I) in which R₅, R₆=alkyl inEtOH was treated with 1 N LiOH and then stirred at room temperatureovernight. The solvent was evaporated under reduced pressure, and theresulting residue was treated with 1 N NaOH and then extracted withEt₂O. The remaining aqueous layer was acidified with 1 N HCl to pH˜2-3and then extracted with EtOAc. The combined organic layer was washedwith 0.1 N HCl, dried over MgSO₄, filtered, and concentrated underreduced pressure to afford carboxylic acid compound of formula (I) inwhich R₆=alkyl, R₅═H.

Compounds of formula (I) in which X is N(R₆) as ester prodrugs ofL-tyrosine, a LAT1 (large neutral amino acid transport system)substrate, that penetrate the brain via a transporter mechanism (J. Med.Chem. 2008, 51, 932-936), may be prepared as described in Scheme 2. Forexample, EDCI-mediated esterification of acid compound of formula (I,R₅, R₆═H) with diprotected tyrosine compound of formula (VIII), theproduct of the N-tert-butoxycarbonylation of the commercially availableL-Tyrosine tert-butyl ester (Nucl. Med. Biol. 2011, 38, 53-62), willgive a tyrosine ester compound of formula (IX). Subsequent cleavage ofthe Boc and t-butyl ester groups of compound of formula (IX) upontreatment of with acid such as trifluoroacetic acid or HCl (Greene &Wuts (1999) Protective Groups in Organic Synthesis, pp 404, 617, 3^(rd)Ed.; John Wiley, New York) will afford a tyrosine ester prodrug compoundof formula (I). Similarly, esterification of an acid compound of formula(I) in which R₅, R₆═H with a tyrosine compound of formula (X), theproduct of the Cs₂CO₃ mediated O-alkylation of a diprotected tyrosinecompound of formula (VIII) with 3-bromopropanol in DMF, will produce atyrosine ester compound of formula (XI) which can be convertedaccordingly to a tyrosine ester prodrug compound of formula (I).

Preparation of a compound of formula (XI) in which R₆═H, L=—(CH₂)₃—: Asolution of a carboxylic acid of formula (I, R₅, R₆═H), NiPr₂Et, DMAP,and EDCI in CH₂Cl₂—CHCN (1:1) was treated with a tyrosine compound offormula (X) and then stirred at room temperature overnight. The reactionmixture was then concentrated under reduced pressure, and the resultingresidue was diluted with water and extracted with CH₂Cl₂. The combinedorganic layer was washed with aq. NaHCO₃, brine, dried over Na₂SO₄,filtered, and concentrated under reduced pressure to give the crudeproduct. Purification by flash chromatography on silica gel withHex:EtOAc afforded a tyrosine ester compound of formula (XI) in whichR₆═H, L=—(CH₂)₃—.

Preparation of a compound of formula (IX) in which R₆═H: A compound offormula (IX) in which R₆═H can be prepared according to the methoddescribed above for the synthesis of a compound of formula (XI) in whichR₆═H, L=—(CH₂)₃—, starting from a carboxylic acid of formula (I) inwhich R₅, R₆═H and a diprotected tyrosine compound of formula (VIII).

Preparation of a tyrosine ester prodrug compound of formula (I) in whichR₆═H, L=—(CH₂)₃—: A solution of tyrosine ester compound of formula (XI)in which R₆═H, L=—(CH₂)₃— in CHCl₃ (or Dioxane) was treated withtrifluoroacetic acid (or 4 M HCl in Dioxane). The reaction mixture wasstirred at room temperature until TLC indicated the consumption ofcompound of formula (XI), and the solvent was then evaporated underreduced pressure to give the crude product. Purification by reversephase preparative HPLC with an CH₃CN—H₂O (0.1% TFA) solution as theeluent afforded the tyrosine ester compound of formula (I) in whichR₆═H, L=—(CH₂)₃—.

Preparation of a tyrosine ester prodrug compound of formula (I) in whichR₆═H: A tyrosine ester prodrug compound of formula (I) in which R₆═H canbe prepared according to the method described above for the synthesis ofcompound of formula (I) in which R₆═H, L=—(CH₂)₃—, starting from acompound of formula (IX, R₆═H).

Compounds of formula (I) in which X is N(R₆), R₄═CF₃, R₆═H, CH₃, andR₅=alkyl, H may be prepared as described in Scheme 3. A series oftrifluoromethylated pyrimidine compounds of formula (I) can result aswell from the β-ketoester compound of formula (XII) in a similar manner.

A compound of formula (XIII) can be prepared by alkylation of acommercially available β-ketoester of formula (XII) with a suitable baseand solvent such as NaH and THF, according to: Aubert, C.; Bégué, J.-P.;Charpentier-Morize, M.; Nee, G.; Langlois. B. J. Fluorine Chem. 1989,44, 3, 361-376.

A compound of formula (XIV) can be prepared according to the methoddescribed for the preparation of a compound of formula (V) as describedin Scheme 1.

A compound of formula (XV) can be prepared according to the methoddescribed for the preparation of a compound of formula (VI) as describedin Scheme 1.

A compound of formula (I) in which R₄═CF₃, R₆═H, and R₅=alkyl can beprepared according to the method described for the preparation of anester compound of formula (I) in which R₆ ═H, R₅=alkyl as described inScheme 1.

A compound of formula (I) in which R₄═CF₃, R₅, R₆═H can be preparedaccording to the method described for the preparation of a carboxylicacid compound of formula (I) in which R₅, R₆═H as described in Scheme 1.

A compound of formula (I) in which R₄═CF₃, R₆═CH₃, R₅=alkyl can beprepared according to the method described for the preparation of anester compound of formula (I) in which R₆=alkyl, R₅=alkyl as describedin Scheme 1.

A compound of formula (I) in which R₄═CF₃, R₆═CH₃, R₅═H can be preparedaccording to the method described for the preparation of a carboxylicacid compound of formula (I) in which R₆ alkyl, R₅═H as described inScheme 1.

Preferred compounds of formula (I) in which X is O and R₅=alkyl, H maybe prepared as described in Scheme 4. For example, treatment of achloropyrimidine compound of formula (VI) with hydroxybenzoate compoundof formula (XVII) in the presence of a base such as Cs₂CO₃ will givephenoxypyrimidine compound of formula (I, R₅=alkyl), which can undergoan ester hydrolysis under basic conditions to give phenoxypyrimidineacid compound of formula (I) in which R₅═H.

A compound of formula (I) in which R₅=alkyl) can be prepared accordingto the following exemplary procedure: A solution of a compound offormula (VI) and a compound of formula (XVII) in DMF was treated withCs₂CO₃. The reaction mixture was stirred at room temperature overnightand then diluted with water and extracted with EtOAc. The aqueous phasewas extracted with EtOAc, and the combined organic layer was washed withbrine, dried over Na₂SO₄, and concentrated under reduced pressure togive the crude product. Purification by flash chromatography on silicagel with Hex:EtOAc afforded the ester compound of formula (I) in whichR₅=alkyl.

A compound of formula (I) in which R₅═H can be prepared according to thefollowing exemplary procedure: A solution of an ester of formula (I) inwhich R₅=alkyl in EtOH was treated with 1 N LiOH and then stirred atroom temperature overnight. The solvent was evaporated under reducedpressure, and the resulting residue was treated with 1 N NaOH and thenextracted with Et₂O. The remaining aqueous layer was acidified with 1 NHCl to pH˜2-3 and then extracted with EtOAc. The combined organic layerwas washed with 0.1 N HCl, dried over Na₂SO₄, filtered, and concentratedunder reduced pressure to afford carboxylic acid compound of formula (I)in which R₅═H. Preferred pyrrolopyrimidine compounds of formula (I),wherein R₅═H, alkyl, R₁₀, R₁₁═H, alkyl, aryl, may be prepared asdescribed in Scheme 5. For example, bromination of the double bond of apyrimidine compound of formula (I) in which X is NH and R₃ is anoptionally substituted allyl group will result in dibromo compound offormula (XVIII) which upon treatment with base can undergo anintramolecular cyclization to afford a pyrrolopyrimidine compound offormula (I) (Khim. Geterotsikl. 1982, 1686-1689; J. Org. Chem. 1991, 56,980-983).

Preparation of a compound of formula (XVIII): To a solution of anappropriately substituted compound of formula (I) in a solvent such asCHCl₃ (or AcOH), wherein R₅═H, alkyl and R₃ is an optionally substitutedallyl group (R₁₀, R₁₁═H, alkyl, aryl), bromine was added dropwise as asolution in CHCl₃ (or AcOH). The reaction mixture was stirred at roomtemperature for 30 minutes, and the solvent was then evaporated to givethe crude dibromo compound of formula (XVIII).

Preparation of a pyrrolopyrimidine compound of formula (I): To asolution of a crude compound of formula (XVIII) in EtOH, a solution of5% KOH in EtOH was added, and the reaction mixture was set up to refluxfor 5 h. The solvent was then evaporated under reduced pressure, and theresulting residue was diluted with water and extracted with EtOAc. Thecombined organic layer was washed with brine, dried over Na₂SO₄,filtered, and concentrated under reduced pressure to give the crudeproduct. Purification by flash chromatography on silica gel withHex:EtOAc or reverse phase preparative HPLC with an CH₃CN—H₂O (0.1% TFA)solution as the eluent afforded the pyrrolopyrimidine compound offormula (I) in which R₅═H.

Preparation of Individual Compounds

The following examples illustrate methods for the preparation ofindividual compounds of formula (I).

Preparation 1: ethyl 2-(2,2,2-trifluoroacetyl)pent-4-enoate

Ethyl 2-(2,2,2-trifluoroacetyl)pent-4-enoate was prepared according toAubert, C.; Bégué, J.-P.; Charpentier-Morize, M.; Nee, G.; Langlois. B.J. Fluorine Chem. 1989, 44, 3, 361-376. To a suspension of 60% w/w NaHin mineral oil (1.08 g, 27.1 mmol) in 20 mL of dry THF under argonatmosphere at 0° C., a solution of ethyl 4,4,4-trifluoro-3-oxobutanoate(5.0 g, 27.1 mmol) in THF (10 mL) was added dropwise. The mixture wasstirred at 0° C. for 1 h, and the solvent was then evaporated underreduced pressure to give a white solid which was suspended in acetone(15 mL) followed by treatment with KI (449 mg, 2.7 mmol). The resultingsuspension was stirred at room temperature for 15 min followed bydropwise addition of a solution of allyl bromide (3.3 g, 27.2 mmol) inacetone (10 mL). The reaction mixture was heated at 60° C. for 48 h, andthe solvent was then evaporated under reduced pressure. The resultingresidue was treated with 1 N HCl (50 mL) and then extracted with CH₂Cl₂(2×30 mL). The combined organic layer was washed with water, dried overMgSO₄, filtered, and concentrated under reduced pressure to give thetitle compound (3.3 g, 54%) as an orange liquid, which was used in thenext step without any further purification.

Preparation 2: 5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ol

Ethyl 2-(2,2,2-trifluoroacetyl)pent-4-enoate (Preparation 1) (3.3 g,14.7 mmol) was added to a solution of NaOEt (5.5 mL of 21% w/w solutionin EtOH, 16.7 mmol) and benzamidine hydrochloride hydrate (2.3 g, 14.7mmol) in EtOH (15 mL). The reaction mixture was stirred overnight underreflux and concentrated under reduced pressure. The resulting residuewas treated with 1 N HCl (40 mL) and then extracted with CH₂Cl₂ (2×30mL). The combined organic layer was washed with 1 N HCl, brine, driedover MgSO₄, filtered, and concentrated under reduced pressure to givethe crude product. Purification by fractional recrystallization inethanol afforded the title compound (2.7 g, 66%) as white needlecrystals. ¹H NMR (250 MHz, CDCl₃) δ 8.27 (d, 2H, J=8.0 Hz), 7.58 (m,3H), 5.92 (m, 1H), 5.21 (dd, 1H, J=1.3, 17.0 Hz), 5.10 (dd, 1H, J=1.5,10.0 Hz), 3.51 (m, 2H). ¹³C NMR (62.9 MHz, CDCl₃) δ 165.3, 155.1, 149.7(q, J_(C-F)=34.3 Hz), 133.5, 132.8, 131.2, 129.3, 127.8, 124.6, 121.7(d, J_(C-F)=276.9 Hz), 117.3, 29.3 (d, J_(C-F)=2.0 Hz). HRMS (ESI-LTQ)for C₁₄H₁₂F₃N₂O [M+H]: calcd, 281.0896; found, 281.0893.

Preparation 3: 5-allyl-4-chloro-2-phenyl-6-(trifluoromethyl)pyrimidine

5-Allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ol (Preparation 2) (2.7g, 9.6 mmol) was dissolved in neat POCl₃ (10 mL), and the resultingsolution was stirred for 6 h under reflux. The solvent was evaporatedunder reduced pressure, and the resulting residue was dissolved in EtOAc(60 mL) and then washed with saturated Na₂CO₃ (2×25 mL) and brine (25mL). The organic layer was dried over MgSO₄, filtered, and concentratedunder reduced pressure to give the crude product. Purification by flashchromatography on silica gel with Hex:EtOAc (10:1) afforded the titlecompound (2.5 g, 87%) as a yellow oil. ¹H NMR (250 MHz, CDCl₃) δ 8.47(dd, 2H, J=1.8, 7.3 Hz), 7.50 (m, 3H), 5.92 (m, 1H), 5.22 (d, 1H, J=11.5Hz), 5.15 (d, 1H, J=18.3 Hz), 3.69 (d, 2H, J=6.0 Hz). ¹³C NMR (62.9 MHz,CDCl₃) δ 164.9, 162.9, 154.8 (q, J_(C-F)=34.5 Hz), 135.0, 132.2, 132.1,128.8, 128.7, 127.3, 121.1 (d, J_(C-F)=277.4 Hz), 118.1, 32.1 (d,J_(C-F)=2.1 Hz). HRMS (ESI-LTQ) for C₁₄H₁₁ClF₃N₂ [M+H]: calcd, 299.0557;299.0560.

Preparation 4: ethyl 2-acetylpent-4-enoate

Ethyl 2-acetylpent-4-enoate was prepared by alkylation of ethyl3-oxobutanoate with a suitable base and solvent by application ofexisting literature protocols: (a) Zhang, Y.; Raines, A. J.; Flowers, R.A. Org. Lett. 2003, 5, 2363-2365. (b) Barbry, D.; Faven, C.; Ajana, A.Org. Prep. Procedure Int. 1994, 26, 469 (c) Nakamura, K.; Miyai, T.;Nagar, A.; Oka, S.; Ohno, A. Bull. Chem. Soc. Jpn. 1989, 62, 1179-1187.

Preparation 5: ethyl 2-acetylpentanoate

Ethyl 2-acetylpentanoate was prepared by alkylation of ethyl3-oxobutanoate with a suitable base and solvent by application ofexisting literature protocol: Nakamura, K.; Miyai, T.; Nagar, A.; Oka,S.; Ohno, A. Bull. Chem. Soc. Jpn. 1989, 62, 1179-1187.

Preparation 6: ethyl 2-acetyl-3-methylbutanoate

Ethyl 2-acetyl-3-methylbutanoate was prepared by alkylation of ethyl3-oxobutanoate with a suitable base and solvent by application ofexisting literature protocol: Beddow, J. E.; Davies, S. G.; Ling, K. B.;Roberts, P. M.; Russel, A. J.; Smith, A. D. Org. Biomol. Chem. 2007, 5,2812-2825.

Preparation 7: ethyl 2-ethyl-3-oxobutanoate

Ethyl 2-ethyl-3-oxobutanoate was prepared by alkylation of ethyl3-oxobutanoate with a suitable base and solvent by application ofexisting literature protocols: (a) Beddow, J. E.; Davies, S. G.; Ling,K. B.; Roberts, P. M.; Russel, A. J.; Smith, A. D. Org. Biomol. Chem.2007, 5, 2812-2825. (b) Nakamura, K.; Miyai, T.; Nagar, A.; Oka, S.;Ohno, A. Bull. Chem. Soc. Jpn. 1989, 62, 1179-1187.

Preparation 8: 5-allyl-6-methyl-2-phenylpyrimidin-4-ol

5-allyl-6-methyl-2-phenylpyrimidin-4-ol was isolated in 78% yield as awhite solid according to the procedure described in Preparation 2,starting from the compound of Preparation 4 and benzamidinehydrochloride hydrate. Spectroscopic data were identical to thosereported in literature. For experimental details and analytical data,see: Kaïm, L. E.; Grimaud, L.; Oble, J. J. Org. Chem. 2007, 72,5835-5838.

Preparation 9: 6-methyl-2-phenyl-5-propylpyrimidin-4-ol

6-methyl-2-phenyl-5-propylpyrimidin-4-ol was isolated in 45% yield as awhite solid according to the procedure described in Preparation 2,starting from the compound of Preparation 5 and benzamidinehydrochloride hydrate.

Preparation 10: 5-isopropyl-6-methyl-2-phenylpyrimidin-4-ol

5-isopropyl-6-methyl-2-phenylpyrimidin-4-ol was isolated in 29% yield asa white solid according to the procedure described in Preparation 2,starting from the compound of Preparation 6 and benzamidinehydrochloride hydrate.

Preparation 11: 5-ethyl-6-methyl-2-phenylpyrimidin-4-ol

5-ethyl-6-methyl-2-phenylpyrimidin-4-ol was isolated in 85% yield as awhite solid according to the procedure described in Preparation 2,starting from the compound of Preparation 7 and benzamidinehydrochloride hydrate.

Preparation 12: 5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ol

5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ol was isolated in 62%yield as a white solid according to the procedure described inPreparation 2, starting from the compound of Preparation 4 and4-chlorobenzamidine hydroiodide.

Preparation 13: 2-(4-chlorophenyl)-6-methyl-5-propylpyrimidin-4-ol

2-(4-chlorophenyl)-6-methyl-5-propylpyrimidin-4-ol was isolated in 59%yield as a white solid according to the procedure described inPreparation 2, starting from the compound of Preparation 5 and4-chlorobenzamidine hydroiodide.

Preparation 14: 2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ol

2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ol was isolated in 33%yield as a white solid according to the procedure described inPreparation 2, starting from the compound of Preparation 7 and4-chlorobenzamidine hydroiodide.

Preparation 15:5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-ol

5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-oI wasisolated in 62% yield as a white solid according to the proceduredescribed in Preparation 2, starting from the compound of Preparation 1and 4-chlorobenzamidine hydroiodide.

Preparation 16: 6-methyl-2-phenylpyrimidin-4-ol

6-methyl-2-phenylpyrimidin-4-ol was prepared from the condensation ofethyl 3-oxobutanoate and benzamidine hydrochloride hydrate according tothe procedure described in Preparation 2. It was isolated in 30% yieldas a white solid. Spectroscopic data were identical to those reported inliterature. For experimental details and analytical data, see: (a) Sun,Q.; Suzenet, F.; Guillaumet, G. J. Org. Chem. 2010, 75, 3473-3476. (b)Zanatta, N.; Fantinel, L.; Lourega, R. V.; Bonacorso, H. G.; Martins, M.A. P. Synthesis 2008, 358-362.

Preparation 17: 5-allyl-4-chloro-6-methyl-2-phenylpyrimidine

5-allyl-4-chloro-6-methyl-2-phenylpyrimidine was isolated in 59% yieldas a beige solid according to the procedure described in Preparation 3,starting from the compound of Preparation 8.

Preparation 18: 4-chloro-6-methyl-2-phenyl-5-propylpyrimidine

4-chloro-6-methyl-2-phenyl-5-propylpyrimidine was isolated in 75% yieldas a beige solid according to the procedure described in Preparation 3,starting from the compound of Preparation 9.

Preparation 19: 4-chloro-5-isopropyl-6-methyl-2-phenylpyrimidine

4-chloro-5-isopropyl-6-methyl-2-phenylpyrimidine was isolated in 81%yield as a beige solid according to the procedure described inPreparation 3, starting from the compound of Preparation 10.

Preparation 20: 4-chloro-5-ethyl-6-methyl-2-phenylpyrimidine

4-chloro-5-ethyl-6-methyl-2-phenylpyrimidine was isolated in 62% yieldas a beige solid according to the procedure described in Preparation 3,starting from the compound of Preparation 11.

Preparation 21: 5-allyl-4-chloro-2-(4-chlorophenyl)-6-methylpyrimidine

5-allyl-4-chloro-2-(4-chlorophenyl)-6-methylpyrimidine was isolated in66% yield as a beige solid according to the procedure described inPreparation 3, starting from the compound of Preparation 12.

Preparation 22: 4-chloro-2-(4-chlorophenyl)-6-methyl-5-propylpyrimidine

4-chloro-2-(4-chlorophenyl)-6-methyl-5-propylpyrimidine was isolated in72% yield as a beige solid according to the procedure described inPreparation 3, starting from the compound of Preparation 13.

Preparation 23: 4-chloro-2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidine

4-chloro-2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidine was isolated in70% yield as a beige solid according to the procedure described inPreparation 3, starting from the compound of Preparation 14.

Preparation 24:5-allyl-4-chloro-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidine

5-allyl-4-chloro-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidine wasisolated in 76% yield as a beige solid according to the proceduredescribed in Preparation 3, starting from the compound of Preparation15.

Preparation 25: 4-chloro-6-methyl-2-phenylpyrimidine

4-chloro-6-methyl-2-phenylpyrimidine was isolated in 59% yield as abeige solid according to the procedure described in Preparation 3,starting from the compound of Preparation 16. For experimental detailsand analytical data, see: Honma, Y.; Sekine, Y.; Hashiyama, T.; Takeda,M.; Ono, Y.; Tsuzurahara, K. Chem. Pharm. Bull. 1982, 30, 4314-4324.

Preparation 26: 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid

4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid wasisolated in 90% yield as a white solid according to the proceduredescribed in Example 3, starting from the compound of Example 6 orExample 7.

Preparation 27: (S)-tert-butyl2-(tert-butoxycarbonylamino)-3-(4-hydroxyphenyl)propanoate

To a solution of commercially available L-tyrosine tert-butyl ester (700mg, 2.95 mmol) in 50 mL of Dioxane-H₂O (5:1), NEt₃ (1.23 mL, 8.85 mmol)and (Boc)₂O (966 mg, 4.42 mmol) were added, and the reaction mixture wasstirred at room temperature. When TLC indicated the consumption ofL-tyrosine tert-butyl ester, the reaction mixture was concentrated underreduced pressure to remove dioxane, and then the resulting solution wasdiluted with water and extracted with EtOAc (2×50 mL). The combinedorganic layer was washed with brine, dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to give the crude product.Purification by flash chromatography on silica gel with MeOH:CH₂Cl₂(1:20) afforded the title compound as a white solid (846 mg, 85%). Formore details and analytical data, see: Wang, L.; Qu, W.; Lieberman, B.P.; Plössl, K.; Kung, H. F. Nucl. Med. Biol. 2011, 38, 53-62)

Preparation 28: (S)-tert-butyl2-(tert-butoxycarbonylamino)-3-(4-(3-hydroxypropoxy)phenyl)propanoate

A solution of (S)-tert-butyl2-(tert-butoxycarbonylamino)-3-(4-hydroxyphenyl)propanoate (Preparation27) (200 mg, 0.592 mmol) in DMF (10 mL) was treated with Cs₂CO₃ (386 mg,1.18 mmol) and 3-bromopropanol (80 μL, 0.89 mmol), and the reactionmixture was stirred at room temperature. When TLC indicated theconsumption of (S)-tert-butyl2-(tert-butoxycarbonylamino)-3-(4-hydroxyphenyl)propanoate, the reactionmixture was diluted with water and extracted with EtOAc (2×30 mL). Thecombined organic layer was washed with brine, dried over Na₂SO₄,filtered, and concentrated under reduced pressure to give the crudeproduct. Purification by flash chromatography on silica gel withHex:EtOAc (1:2) afforded the title compound as a white solid (164 mg,70%).

Preparation 29:(S)-4-(3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-oxopropyl)phenyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate

A solution of 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid (Preparation 26) (100 mg, 0.29 mmol), NEt₃ (81 μL, 0.58 mmol), DMAP(7 mg, 0.058 mmol) and EDCI (68 mg, 0.43 mmol) in 10 mL of CH₂Cl₂—CH₃CN(1:1) was treated with (S)-tert-butyl2-(tert-butoxycarbonylamino)-3-(4-hydroxyphenyl)propanoate (Preparation27) (147 mg, 0.43 mmol) and the reaction mixture was stirred at roomtemperature. When TLC indicated the consumption of4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid, thereaction mixture was concentrated under reduced pressure to remove thesolvent, and then the resulting residue was diluted with water andextracted with EtOAc (2×30 mL). The combined organic layer was washedwith brine, dried over Na₂SO₄, filtered and concentrated under reducedpressure to give the crude product. Purification by flash chromatographyon silica gel with Hex:EtOAc (1:6) afforded the title compound as awhite solid (83 mg, 43%).

Preparation 30:(S)-3-(4-(3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-oxopropyl)phenoxy)propyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate

A solution of 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid (Preparation 26) (100 mg, 0.29 mmol), NEt₃ (81 μL, 0.58 mmol), DMAP(7 mg, 0.058 mmol), and EDCI (68 mg, 0.43 mmol) in 10 mL of CH₂Cl₂—CH₃CN(1:1) was treated with (S)-tert-butyl2-(tert-butoxycarbonylamino)-3-(4-(3-hydroxypropoxy)phenyl)propanoate(Preparation 28) (172 mg, 0.43 mmol) and the reaction mixture wasstirred at room temperature. When TLC indicated the consumption of4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid, thereaction mixture was concentrated under reduced pressure to remove thesolvent, and then the resulting residue was diluted with water andextracted with EtOAc (2×30 mL). The combined organic layer was washedwith brine, dried over Na₂SO₄, filtered, and concentrated under reducedpressure to give the crude product. Purification by flash chromatographyon silica gel with Hex:EtOAc (1:6) afforded the title compound as awhite solid (75 mg, 36%).

Example 1: ethyl4-(5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoate

A solution of 5-allyl-4-chloro-2-phenyl-6-(trifluoromethyl)pyrimidine(Preparation 3) (2.5 g, 8.37 mmol), ethyl 4-aminobenzoate (2.5 g, 15.1mmol), and a catalytic amount of concentrated HCl (2-3 drops) in EtOH(15 mL) was stirred under reflux. The reaction progress was monitored byTLC, and catalytic amounts of concentrated HCl were gradually added tothe reaction mixture in order to drive the reaction to completion. WhenTLC indicated the consumption of5-allyl-4-chloro-2-phenyl-6-(trifluoromethyl)pyrimidine, the solvent wasevaporated under reduced pressure. The resulting residue was treatedwith water (60 mL) followed by extraction with EtOAc (3×40 mL). Thecombined organic layer was washed with water (3×25 mL), dried overMgSO₄, filtered, and concentrated under reduced pressure to give thecrude product. Purification by flash chromatography on silica gel withHex:EtOAc (10:1 to 6:1) afforded the title compound (1.1 g, 31%) as ayellowish solid. ¹H NMR (250 MHz, CDCl₃) δ 8.44 (m, 2H), 8.11 (d, 2H,J=8.8 Hz), 7.74 (d, 2H, J=8.8 Hz), 7.50 (m, 3H), 7.23 (s, 1H), 6.0 (m,1H), 5.42 (dd, 1H, J=0.8, 10.0 Hz), 5.37 (d, 1H, J=16.8 Hz), 4.40 (q,2H, J=7.1 Hz), 3.60 (d, 2H, J=5.5 Hz), 1.43 (t, 3H, J=7.1 Hz). ¹³C NMR(62.9 MHz, CDCl₃) δ 166.3 (C), 162.3 (C), 160.3 (C), 152.8 (C, q,J_(C-F)=32.9 Hz), 142.8 (C), 136.8 (C), 133.6 (CH), 131.2 (CH), 130.8(CH), 128.7 (CH), 128.4 (CH), 125.6 (C), 121.8 (C, d, J_(C-F)=276.9 Hz),119.8 (CH), 118.9 (═CH₂), 111.9 (C), 61.0 (CH₂), 30.1 (CH₂, d,J_(C-F)=2.1 Hz), 14.5 (CH₃). HRMS (ESI-LTQ) for C₂₃H₂₁F₃N₃O₂ [M+H]:calcd, 428.1580; found, 428.1583.

Example 2: ethyl4-((5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-yl)(methyl)amino)benzoate

A solution of ethyl4-(5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoate(Example 1)(1.1 g, 2.58 mmol) in DMF (10 mL) was treated with Cs₂CO₃(1.0 g, 3.07 mmol) and CH₃1 (241 μL, 3.87 mmol) and stirred at roomtemperature overnight. The reaction mixture was then diluted with water(40 mL) and extracted with EtOAc (2×25 mL). The combined organic layerwas washed with water (2×20 mL), dried over MgSO₄, filtered, andconcentrated under reduced pressure to give the crude product.Purification by flash chromatography on silica gel with Hex:EtOAc (10:1to 6:1) afforded the title compound (1.0 g, 88%) as a yellow solid. ¹HNMR (250 MHz, CDCl₃) δ 8.49 (m, 2H), 8.02 (d, 2H J=8.8 Hz), 7.50 (m,3H), 7.01 (d, 2H, J=8.8 Hz), 5.56 (m, 1H), 4.94 (dd, 1H, J=1.5, 10.0Hz), 4.75 (dd, 1H, J=1.5, 17.0 Hz), 4.38 (q, 2H, J=7.1 Hz), 3.65 (s,3H), 3.05 (d, 2H, J=6.0 Hz), 1.42 (t, 3H, J=7.1 Hz). ¹³C NMR (62.9 MHz,CDCl₃) δ 166.0 (C), 165.5 (C), 162.3 (C), 154.8 (C, q, J_(C-F)=32.9 Hz),150.7 (CH), 136.6 (C), 133.9 (CH), 131.4 (CH), 131.3 (CH), 128.7 (CH),128.4 (CH), 126.1 (C), 121.8 (C, d, J_(C-F)=277.1 Hz), 121.1 (CH), 120.3(C), 116.6 (═CH₂), 61.1 (CH₂), 41.1 (CH₃), 30.1 (CH₂, d, J_(C-F)=2.3Hz), 14.5 (CH₃). HRMS (ESI-LTQ) for C₂₄H₂₃F₃N₃O₂ [M+H]: calcd, 442.1737;found, 442.1740.

Example 3:4-((5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-yl)(methyl)amino)benzoicacid

A solution of ethyl4-((5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-yl)(methyl)amino)benzoate(Example 2) (1.0 g, 2.27 mmol) in 20 mL of EtOH was treated with 1 NLiOH (5 mL, 5 mmol) and then stirred at room temperature overnight. Thesolvent was evaporated under reduced pressure, and the resulting residuewas treated with 1 N NaOH (50 mL) and then extracted with Et₂O (2×20mL). The remaining aqueous layer was acidified with 1 N HCl (40 mL) topH˜2-3 and then extracted with EtOAc (3×30 mL). The combined organiclayer was washed with 0.1 N HCl (20 mL), dried over MgSO₄, filtered, andconcentrated under reduced pressure to afford the title compound (800mg, 85%) as an off-white solid in high purity. The title compound can befurther recrystallized in ethanol. ¹H NMR (250 MHz, CDCl₃) δ 8.47 (m,2H), 8.05 (d, 2H, J=8.8 Hz), 7.47 (m, 3H), 6.98 (d, 2H, J=8.8 Hz), 5.55(m, 1H), 4.93 (dd, 1H, J=1.0, 10.0 Hz), 4.72 (dd, J=0.9, 17.1 Hz), 3.62(s, 3H), 3.08 (d, 2H, J=5.8 Hz). ¹³C NMR (62.9 MHz, CDCl₃) δ 171.7 (C),165.6 (C), 162.6 (C), 155.1 (C, q, J_(C-F)=32.9 Hz), 151.5 (C), 136.5(C), 133.9 (CH), 132.1 (CH), 131.4 (CH), 128.8 (CH), 128.4 (CH), 124.1(C), 121.8 (C, d, J_(C-F)=277.2 Hz), 121.1 (C), 120.2 (CH), 116.7 (═CHO,40.8 (CH₃), 30.6 (CH₂, d, J_(C-F)=2.1 Hz). HRMS (ESI-LTQ) forC₂₂H₁₉F₃N₃O₂ [M+H]: calcd, 414.1424; found, 414.1418.

Example 4:4-(5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoic acid

4-(5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoic acidwas isolated in 65% yield as a white solid according to the proceduredescribed in Example 3, starting from the compound of Example 1. It wasisolated in a mixture with the isomeric compound of Example 5 (Example4/Example 5=55/45).

Example 5:(E)-4-(2-phenyl-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoicacid

(E)-4-(2-phenyl-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoicacid was isolated as a white solid according to the procedure describedin Example 3, starting from the compound of Example 1. It was isolatedin a mixture with the compound of Example 4 (Example 4/Example 5=55/45).

Example 6: ethyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate

Ethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate wasisolated in 45% yield as a white solid according to the proceduredescribed in Example 1, starting from the compound of Preparation 17 andethyl 4-aminobenzoate.

Example 7: methyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate

Methyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate wasisolated in 45% yield as a white solid according to the proceduredescribed in Example 1, starting from the compound of Preparation 17 andmethyl 4-aminobenzoate.

Example 8: ethyl4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoate

Ethyl 4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoatewas isolated in 56% yield as a white solid according to the proceduredescribed in Example 2, starting from the compound of Example 6 andiodomethane.

Example 9: methyl4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoate

Methyl4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoate wasisolated in 58% yield as a white solid according to the proceduredescribed in Example 2, starting from the compound of Example 7 andiodomethane.

Example 10:4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoic acid

4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoic acidwas isolated in 63% yield as a white solid according to the proceduredescribed in Example 3, starting from the compound of Example 8 orExample 9.

Example 11: 4-(6-methyl-2-phenyl-5-propylpyrimidin-4-ylamino)benzoicacid

4-(6-methyl-2-phenyl-5-propylpyrimidin-4-ylamino)benzoic acid wasprepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 18 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(6-methyl-2-phenyl-5-propylpyrimidin-4-ylamino)benzoate to give thetitle compound as a beige solid (36% yield for two steps).

Example 12: 4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid

4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid wasprepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 19 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate to give thetitle compound as a beige solid (41% yield for two steps).

Example 13: 4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid

4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid wasprepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 20 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate to give thetitle compound as a beige solid (33% yield for two steps).

Example 14:4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoic acid

4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoic acidwas prepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 21 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoate togive the title compound as a beige solid (44% yield for two steps).

Example 15:4-(2-(4-chlorophenyl)-6-methyl-5-propylpyrimidin-4-ylamino)benzoic acid

4-(2-(4-chlorophenyl)-6-methyl-5-propylpyrimidin-4-ylamino)benzoic acidwas prepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 22 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(2-(4-chlorophenyl)-6-methyl-5-propylpyrimidin-4-ylamino)benzoate togive the title compound as a white solid (45% yield for two steps).

Example 16:4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ylamino)benzoic acid

4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ylamino)benzoic acidwas prepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 23 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ylamino)benzoate togive the title compound as a white solid (50% yield for two steps).

Example 17:4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-2-fluorobenzoic acid

4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-2-fluorobenzoic acidwas prepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 17 and methyl4-amino-2-fluorobenzoate followed by ester hydrolysis of the resultingethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-2-fluorobenzoateto give the title compound as a white solid (46% yield for two steps).

Example 18:4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-3-methylbenzoic acid

4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-3-methylbenzoic acidwas prepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 17 and methyl4-amino-3-methylbenzoate followed by ester hydrolysis of the resultingmethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-3-methylbenzoateto give the title compound as a beige solid (35% yield for two steps).

Example 19:4-(5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoicacid

4-(5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoicacid was prepared according to the procedures described in Examples 1and 3, employing the coupling of compound of Preparation 24 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoateto give the title compound as a white solid (25% yield for two steps).It was isolated in a mixture with the isomeric compound of Example 20(Example 19/Example 20=60/40).

Example 20:(E)-4-(2-(4-chlorophenyl)-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoicacid

(E)-4-(2-(4-chlorophenyl)-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoicacid was prepared according to the procedures described in Examples 1and 3, employing the coupling of compound of Preparation 24 and ethyl4-aminobenzoate followed by ester hydrolysis of the resulting ethyl4-(5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoateto give the title compound as a white solid (25% yield for two steps).It was isolated in a mixture with the compound of Example 19 (Example19/Example 20=60/40).

Example 21: 3-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid

3-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid wasprepared according to the procedures described in Examples 1 and 3,employing the coupling of compound of Preparation 17 and ethyl3-aminobenzoate followed by ester hydrolysis of the resulting ethyl3-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate to give thetitle compound as a beige solid (28% yield for two steps).

Example 22:3-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoic acid

3-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoic acidwas prepared according to the procedures described in Examples 1, 2, and3, employing the coupling of compound of Preparation 17 and ethyl3-aminobenzoate to give ethyl3-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate. SubsequentN-methylation resulted in ethyl3-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)benzoate whichunderwent an ester hydrolysis to give the title compound as a whitesolid (28% yield for three steps).

Example 23:4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-2-fluorobenzoicacid

4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-2-fluorobenzoicacid was prepared according to the procedures described in Examples 1,2, and 3, employing the coupling of compound of Preparation 17 andmethyl 4-amino-2-fluorobenzoate to give methyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-2-fluorobenzoate.Subsequent N-methylation resulted in methyl4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-2-fluorobenzoatewhich underwent an ester hydrolysis to give the title compound as abeige solid (35% yield for three steps).

Example 24:4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-3-methylbenzoicacid

4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-3-methylbenzoicacid was prepared according to the procedures described in Examples 1,2, and 3, employing the coupling of compound of Preparation 17 andmethyl 4-amino-3-methylbenzoate to give methyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)-3-methylbenzoate.Subsequent N-methylation resulted in methyl4-((5-allyl-6-methyl-2-phenylpyrimidin-4-yl)(methyl)amino)-3-methylbenzoatewhich underwent an ester hydrolysis to give the title compound as awhite solid (30% yield for three steps).

Example 25:4-((5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yl)(methyl)amino)benzoicacid

4-((5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yl)(methyl)amino)benzoicacid was prepared according to the procedures described in Examples 1,2, and 3, employing the coupling of compound of Preparation 21 and ethyl4-aminobenzoate to give ethyl4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-ylamino)benzoate.Subsequent N-methylation resulted in ethyl4-((5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yl)(methyl)amino)benzoatewhich underwent an ester hydrolysis to give the title compound as awhite solid (35% yield for three steps).

Example 26:4-((2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yl)(methyl)amino)benzoicacid

4-((2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yl)(methyl)amino)benzoicacid was prepared according to the procedures described in Examples 1,2, and 3, employing the coupling of compound of Preparation 23 and ethyl4-aminobenzoate to give ethyl4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-ylamino)benzoate.Subsequent N-methylation resulted in ethyl4-((2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yl)(methyl)amino)benzoatewhich underwent an ester hydrolysis to give the title compound as awhite solid (32% yield for three steps).

Example 27: 4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid

4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid was preparedaccording to the procedures described in Examples 1 and 3, employing thecoupling of compound of Preparation 25 and ethyl 4-aminobenzoatefollowed by ester hydrolysis of the resulting ethyl4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoate to give the titlecompound as a white solid (48% yield for two steps).

Example 28: 4-(allyl(6-methyl-2-phenylpyrimidin-4-yl)amino)benzoic acid

4-(allyl(6-methyl-2-phenylpyrimidin-4-yl)amino)benzoic acid was preparedaccording to the procedures described in Examples 1, 2, and 3, employingthe coupling of compound of Preparation 25 and ethyl 4-aminobenzoate togive ethyl 4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoate. SubsequentN-alkylation with allyl bromide resulted in ethyl4-(allyl(6-methyl-2-phenylpyrimidin-4-yl)amino)benzoate which underwentan ester hydrolysis to give the title compound as a white solid (29%yield for three steps).

Example 29: 4-((6-methyl-2-phenylpyrimidin-4-yl)(propyl)amino)benzoicacid

4-((6-methyl-2-phenylpyrimidin-4-yl)(propyl)amino)benzoic acid wasprepared according to the procedures described in Examples 1, 2, and 3,employing the coupling of compound of Preparation 25 and ethyl4-aminobenzoate to give ethyl4-(6-methyl-2-phenylpyrimidin-4-ylamino)benzoate. SubsequentN-alkylation with 1-propylbromide resulted in ethyl4-((6-methyl-2-phenylpyrimidin-4-yl)(propyl)amino)benzoate whichunderwent an ester hydrolysis to give the title compound as a whitesolid (32% yield for three steps).

Example 30: pivaloyloxymethyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate

A solution of 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid (Preparation 26) (100 mg, 0.29 mmol) in DMF (5 mL) was treated withCs₂CO₃ (189 mg, 0.58 mmol) and chloromethyl pivalate (50 μL, 0.35 mmol).The reaction mixture was stirred at room temperature until TLC indicatedthe consumption of4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoic acid. It wasthen diluted with water and extracted with EtOAc (2×25 mL). The combinedorganic layer was washed with brine, dried over MgSO₄, filtered, andconcentrated under reduced pressure to give the crude product.Purification by flash chromatography on silica gel with Hex:EtOAc (10:1)afforded the title compound as a white solid (107 mg, 80%).

Example 31:(S)-2-(4-(4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoyloxy)phenyl)-1-carboxyethanaminiumchloride

A solution of(S)-4-(3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-oxopropyl)phenyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate (Preparation29) (50 mg, 0.075 mmol) in dioxane was treated with 4 M HCl in dioxane(187 μL, 0.75 mmol), and the reaction mixture was stirred at roomtemperature. When TLC indicated the consumption of(S)-4-(3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-oxopropyl)phenyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate, the reactionmixture was concentrated under reduced pressure to give the crudeproduct. Purification by reverse phase preparative HPLC with a CH₃CN—H₂O(0.1% TFA) solution (90:10) as the eluent, afforded the title compoundas a white solid (11 mg, 28%).

Example 32:(S)-2-(4-(3-(4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoyloxy)propoxy)phenyl)-1-carboxyethanaminiumchloride

A solution of(S)-3-(4-(3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-oxopropyl)phenoxy)propyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate (Preparation30) (50 mg, 0.069 mmol) in dioxane was treated with 4 M HCl in dioxane(173 μL, 0.69 mmol), and the reaction mixture was stirred at roomtemperature. When TLC indicated the consumption of(S)-3-(4-(3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-oxopropyl)phenoxy)propyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoate, the reactionmixture was concentrated under reduced pressure to give the crudeproduct. Purification by reverse phase preparative HPLC with a CH₃CN—H₂O(0.1% TFA) solution (90:10) as the eluent, afforded the title compoundas a white solid (15 mg, 36%).

Example 33: ethyl4-(5-allyl-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-yloxy)benzoate

A solution of5-allyl-4-chloro-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidine(Preparation 24) (100 mg, 0.30 mmol) in DMF (10 mL) was treated withCs₂CO₃ (196 mg, 0.60 mmol) and ethyl 4-hydroxybenzoate (75 mg, 0.45mmol), and the reaction mixture was stirred at room temperature. WhenTLC indicated the consumption of5-allyl-4-chloro-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidine, thereaction mixture was diluted with water and extracted with EtOAc (2×25mL). The combined organic layer was washed with aq. NaHCO₃, brine, driedover Na₂SO₄, filtered, and concentrated under reduced pressure to givethe crude product. Purification by flash chromatography on silica gelwith Hex:EtOAc (1:6) afforded the title compound in a mixture with theisomeric compound of Example 34 (Example 33/Example 34=1/2). Furtherpurification by reverse phase preparative HPLC with a CH₃CN—H₂O (0.1%TFA) solution (90:10) as the eluent, enabled the isolation of the titlecompound as a white solid (19 mg, 14%).

Example 34: (E)-ethyl4-(2-(4-chlorophenyl)-5-(prop-1-enyl)-6-(trifluoromethyl)pyrimidin-4-yloxy)benzoate

A solution of5-allyl-4-chloro-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidine(Preparation 24) (100 mg, 0.30 mmol) in DMF (10 mL) was treated withCs₂CO₃ (196 mg, 0.60 mmol) and ethyl 4-hydroxybenzoate (75 mg, 0.45mmol), and the reaction mixture was stirred at room temperature. WhenTLC indicated the consumption of5-allyl-4-chloro-2-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidine, thereaction mixture was diluted with water and extracted with EtOAc (2×25mL). The combined organic layer was washed with aq. NaHCO₃, brine, driedover Na₂SO₄, filtered and concentrated under reduced pressure to givethe crude product, Purification by flash chromatography on silica gelwith Hex:EtOAc (1:6) afforded the title compound in a mixture with theisomeric compound of Example 33 (Example 33/Example 34=1/2). Furtherpurification by reverse phase preparative HPLC with a CH₃CN—H₂O (0.1%TFA) solution (90:10) as the eluent, enabled the isolation of compoundof the title compound as a white solid (40 mg, 29%).

Example 35: 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid

4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid was preparedaccording to the procedures described in Examples 33 and 3, employingthe coupling of compound of Preparation 17 and ethyl 4-hydroxybenzoatefollowed by ester hydrolysis of the resulting ethyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoate to give the titlecompound as a white solid (45% yield for two steps). It was isolated ina mixture with the isomeric compound of Example 36 (Example 35/Example36=1/4).

Example 36:(E)-4-(6-methyl-2-phenyl-5-(prop-1-enyl)pyrimidin-4-yloxy)benzoic acid

(E)-4-(6-methyl-2-phenyl-5-(prop-1-enyl)pyrimidin-4-yloxy)benzoic acidwas prepared according to the procedures described in Examples 33 and 3,employing the coupling of compound of Preparation 17 and ethyl4-hydroxybenzoate followed by ester hydrolysis of the resulting ethyl4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoate to give the titlecompound as a white solid (45% yield for two steps). It was isolated ina mixture with the isomeric compound of Example 35 (Example 35/Example36=1/4).

Example 37:4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yloxy)benzoic acid

4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yloxy)benzoic acid wasprepared according to the procedures described in Examples 33 and 3,employing the coupling of compound of Preparation 21 and ethyl4-hydroxybenzoate followed by ester hydrolysis of the resulting ethyl4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yloxy)benzoate to givethe title compound as a white solid (47% yield for two steps). It wasisolated in a mixture with the isomeric compound of Example 38 (Example37/Example 38=1/4).

Example 38:(E)-4-(2-(4-chlorophenyl)-6-methyl-5-(prop-1-enyl)pyrimidin-4-yloxy)benzoicacid

(E)-4-(2-(4-chlorophenyl)-6-methyl-5-(prop-1-enyl)pyrimidin-4-yloxy)benzoicacid was prepared according to the procedures described in Examples 33and 3, employing the coupling of compound of Preparation 21 and ethyl4-hydroxybenzoate followed by ester hydrolysis of the resulting ethyl4-(5-allyl-2-(4-chlorophenyl)-6-methylpyrimidin-4-yloxy)benzoate to givethe title compound as a white solid (47% yield for two steps). It wasisolated in a mixture with the isomeric compound of Example 37. (Example37/Example 38=1/4).

Example 39:4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)-2-fluorobenzoic acid

4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)-2-fluorobenzoic acid wasprepared according to the procedures described in Examples 33 and 3,employing the coupling of compound of Preparation 17 and ethyl2-fluoro-4-hydroxybenzoate followed by ester hydrolysis of the resultingethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)-2-fluorobenzoate togive the title compound as a mixture with the isomeric compound ofExample 40 (Example 39/Example 40=1/2). Further purification by reversephase preparative HPLC with a CH₃CN—H₂O (0.1% TFA) solution (90:10) asthe eluent, enabled the isolation of the title compound as a beige solid(17%).

Example 40:(E)-2-fluoro-4-(6-methyl-2-phenyl-5-(prop-1-enyl)pyrimidin-4-yloxy)benzoicacid

(E)-2-fluoro-4-(6-methyl-2-phenyl-5-(prop-1-enyl)pyrimidin-4-yloxy)benzoicacid was prepared according to the procedures described in Examples 33and 3, employing the coupling of compound of Preparation 17 and ethyl2-fluoro-4-hydroxybenzoate followed by ester hydrolysis of the resultingethyl 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-yloxy)-2-fluorobenzoate togive the title compound in a mixture with the isomeric compound ofExample 39 (Example 39/Example 40=1/2). Further purification by reversephase preparative HPLC with a CH₃CN—H₂O (0.1% TFA) solution (90:10) asthe eluent, enabled the isolation of the title compound as a white solid(34%).

Example 41: 4-(6-methyl-2-phenyl-5-propylpyrimidin-4-yloxy)benzoic acid

4-(6-methyl-2-phenyl-5-propylpyrimidin-4-yloxy)benzoic acid was preparedaccording to the procedures described in Examples 33 and 3, employingthe coupling of compound of Preparation 18 and ethyl 4-hydroxybenzoatefollowed by ester hydrolysis of the resulting ethyl4-(6-methyl-2-phenyl-5-propylpyrimidin-4-yloxy)benzoate to give thetitle compound as a white solid (44% yield for two steps).

Example 42: 4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoicacid

4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid wasprepared according to the procedures described in Examples 33 and 3,employing the coupling of compound of Preparation 19 and ethyl4-hydroxybenzoate followed by ester hydrolysis of the resulting ethyl4-(5-isopropyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoate to give thetitle compound as a beige solid (42% yield for two steps).

Example 43: 4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid

4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoic acid was preparedaccording to the procedures described in Examples 33 and 3, employingthe coupling of compound of Preparation 20 and ethyl 4-hydroxybenzoatefollowed by ester hydrolysis of the resulting ethyl4-(5-ethyl-6-methyl-2-phenylpyrimidin-4-yloxy)benzoate to give the titlecompound as a white solid (44% yield for two steps).

Example 44:4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yloxy)benzoic acid

4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yloxy)benzoic acid wasprepared according to the procedures described in Examples 31 and 3,employing the coupling of compound of Preparation 23 and ethyl4-hydroxybenzoate followed by ester hydrolysis of the resulting ethyl4-(2-(4-chlorophenyl)-5-ethyl-6-methylpyrimidin-4-yloxy)benzoate to givethe title compound as a white solid (42% yield for two steps).

Example 45:4-(4,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzoic acid

A solution of 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid (Preparation 26) (20 mg, 0.058 mmol) in glacial acetic acid (5 mL)was treated with bromine (12 μL, 0.232 mmol) as a solution in aceticacid (0.2 mL). The reaction mixture was stirred at room temperature for1 h and then concentrated under reduced pressure. The resulting residuewas dissolved in EtOH (4 mL), treated with a solution of 5% KOH in EtOH(1 mL) and then refluxed for 5 h. After cooling at room temperature, thesolvent was evaporated under reduced pressure, and the resulting residuewas treated with 1 N NaOH (5 mL) and then extracted with Et₂O (2×20 mL).The remaining aqueous layer was acidified with 1 N HCl to pH˜2-3 andthen extracted with EtOAc (3×30 mL). The combined organic layer wasdried over MgSO₄, filtered, and concentrated under reduced pressure toafford the crude product. Purification by reverse phase preparative HPLCwith a CH₃CN—H₂O (0.1% TFA) solution (90:10) as the eluent, enabled theisolation of the title compound as a beige solid (4 mg, 20%).

Example 46:3-bromo-4-(4,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-cl]pyrimidin-7-yl)benzoicacid

A solution of 4-(5-allyl-6-methyl-2-phenylpyrimidin-4-ylamino)benzoicacid (Preparation 26) (20 mg, 0.058 mmol) in glacial acetic acid (5 mL)was treated with bromine (12 μL, 0.232 mmol) as a solution in aceticacid (200 μL). The reaction mixture was stirred at room temperatureovernight and then concentrated under reduced pressure. The resultingresidue was dissolved in EtOH (4 mL), treated with a solution of 5% KOHin EtOH (1 mL) and then refluxed for 5 h. After cooling at roomtemperature, the solvent was evaporated under reduced pressure, and theresulting residue was treated with 1 N NaOH (5 mL) and then extractedwith Et₂O (2×20 mL). The remaining aqueous layer was acidified with 1 NHCl to pH˜2-3 and then extracted with EtOAc (3×30 mL). The combinedorganic layer was dried over MgSO₄, filtered, and concentrated underreduced pressure to afford the crude product. Purification by reversephase preparative HPLC with a CH₃CN—H₂O (0.1% TFA) solution (90:10) asthe eluent, enabled the isolation of the title compound as a beige solid(7 mg, 27%).

Example 47:4-(6-methyl-2-phenyl-4-(trifluoromethyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)benzoicacid

A solution of ethyl4-(5-allyl-2-phenyl-6-(trifluoromethyl)pyrimidin-4-ylamino)benzoate(Example 1) (20 mg, 0.047 mmol) in glacial acetic acid (5 mL) wastreated with bromine (10 μL, 0.188 mmol) as a solution in acetic acid(200 μL). The reaction mixture was stirred at room temperature for 5 hand then concentrated under reduced pressure. The resulting residue wasdissolved in EtOH (4 mL), treated with a solution of 5% KOH in EtOH (1mL), and then refluxed for 5 h. After cooling at room temperature, theresulting solution was treated with 1 N LiOH (100 μL, 0.1 mmol) and thenstirred at room temperature overnight. The solvent was evaporated underreduced pressure and the resulting residue was treated with 1 N NaOH (5mL) and then extracted with Et₂O (2×20 mL). The remaining aqueous layerwas acidified with 1 N HCl to pH˜2-3 and then extracted with EtOAc (3×30mL). The combined organic layer was dried over MgSO₄, filtered, andconcentrated under reduced pressure to afford the crude product.Purification by reverse phase preparative HPLC with a CH₃CN—H₂O (0.1%TFA) solution (90:10) as the eluent, enabled the isolation of the titlecompound as a beige solid (6 mg, 32%).

II. Nurr1:RXRα-Dependent Transcription-Activating Capabilities ofSynthesized Compounds of the Invention

Synthesized compounds of the invention and their analogs, shown in Table1, below, were evaluated for activity in cellular assays. The in vitrotransactivation assay determined the capacity of the compounds of theinvention to activate the Nurr1:RXR heterodimers. Naive SHSY-5Y cellswere transiently co-transfected, using Lipofectamine 2000 (Invitrogen),with a plasmid expressing the human receptor Nurr1, a plasmid expressingthe RXR human receptor (RXRα or RXRγ receptor) and a reporter plasmidDR5-TK-Luc. After 24 hours, the culture medium was changed. The testcompounds were added (final concentration between 0.5, 2.5 and 12.5 μM)in the culture medium. After incubation overnight, the expression ofluciferase was measured (FIG. 1A). Positive control cells were treatedwith XCT0135908 (1 μM; WO 2005/047268) which induces activation of theNurr1:RXR heterodimer and results in maximal expression of luciferase.The transcription-activating activities of various compounds of theinvention are shown in FIG. 1B, as well as in Table 1, below. In thetransactivation assay, compound 3 exhibited an EC₅₀ value of (0.9 μM)(FIG. 1C). Furthermore, compound 3 is selective for the Nurr1:RXRαheterodimer over the Nurr1:RXRγ heterodimer (FIG. 1D).

TABLE 1 Transcription-activating properties of select compounds of theinvention as assessed in a Nurr1: RXRα-dependent luciferase reporterassay Example Compound EC₅₀ (μM) 1

>50 2

Not tested 3

0.9 4

0.2 (sample contained 45% of the styryl isomer of Example 5) 5

0.3 6

>100 7

Not tested 8

Not tested 9

>100 10

>20 11

1 12

1 13

5 14

2 15

>10 16

2 17

Not tested 18

Not tested 19

10 (sample contained 40% of the styryl isomer of Example 20) 20

>10 21

Not tested 22

Not tested 23

7 24

>100 25

7 26

4 27

Not tested 28

Not tested 29

10 30

2 31

10 32

5 33

Not tested 34

Not tested 35

>100 (sample contained 80% of the styryl isomer of Example 36) 36

>10 37

>100 (sample contained 80% of the styryl isomer of Example 38) 38

Not tested 39

>10 40

Not tested 41

>100 42

1 43

>100 44

>10 45

2.5 46

0.7 47

Not tested

Compound 3 specifically activates Nurr1:RXRα heterodimers over otherRXR-containing transcription co-activator complexes. Specificity assaysfusing DNA sequences of the gal4 DNA binding domain to the nuclearreceptor Ligand-Binding-Domain (LDB) and to vp16 were performed asfollows: Human SHSY-5Y cells were cotransfected with 20 ng ofMH100-tk-luciferase, 10 ng of CMX-bgal, 20 ng of CMX-GAL4-RXRα-LBD and20 ng of CMX-VP16-NR-LBD, where NR stands for either Nurr1, VDR, PPARγ.CMX-VP16-VDR-LBD and CMX-VP16-PPARγ-LBD were kindly provided by DrMakishima (Nikon University School of Medicine). CMX-VP16-Nurr1-LBD wasproduced by cloning the Nurr1 LBD in the CMX-VP16 vector using KpnI.More specifically, Nurr1-LBD was isolated by PCR from a vectorcontaining the Nurr1 cDNA using a set of primers flanking the Nurr1 LBDsequence and carrying at both sides the KpnI restriction sites, namelyNurr1 Forward TGCGGTACCAGGAGCCCTCTCCCCCTTC (SEQ ID NO: 1) and Nurr1Reverse TGCGGTACCTTAGAAAGGTAAAGTGTCCA (SEQ ID NO: 2). To exclude thepossibility that compound 3 might transactivate RXRγ:Nurr1 heterodimers,CMX-GAL4-RXRγ-LBD was constructed by cloning the RXRγ LBD that wasisolated by PCR from a vector containing the RXRγ cDNA (RIKEN, Tokyo,Japan) using the following Forward and Reverse primers carrying KpnI andNheI restriction sites, respectively, namely RXRγ ForwardTGCGGTACCGAGCGAGCTGAGAGTGAG (SEQ ID NO: 3) and RXRγ LBD ReverseTGCGCTAGCTCAGGTGATCTGCAGC (SEQ ID NO: 4). 17 hours after transfectionthe compound was added to the medium at the corresponding concentrationsand left for another 12 hours. Then the cells were assayed forluciferase activity normalized to galactosidase activity.

III. Neuroprotective Effect of Compounds of the Invention in Mice

To assess the neuroprotective effect of compound 3, this compound wasdosed to wt mice intraperitoneally (IP) at 10 mg/kg injections ofcompound 3 in Ethanol:PG:Saline 10:30:60. After 2 h and 4 h animals (n=3per time point) were sacrificed, and their brains obtained. Afterextraction c-jun and tyrosine hydroxylase (TH) levels in brain extractwere determined by qPCR (FIG. 1E, 1F).

IV. Neuroprotective Effect of Compounds of the Invention In Vitro

MPP+, eradicates approximately 75% of the cells in 24 hours. Addition ofcompound 3 approximately doubles the number of surviving cells (FIG. 1C, D, E). Naive Neuro2A cells (FIG. 2A) or SH-SY5Y neuroblastoma cellsFIG. 2B,C) were cultured in RPMI 1640, 10% fetal bovine serum at 37° C.Cells were pretreated with compound 3 for 12-24 h. Subsequent change ofculture medium with addition of 0, 1, 2 and 4 mM MPP+ induced celldeath. MTT assays determined the relative number of surviving cells FIG.2D).

V. SHSY5Y Cells are Protected from H₂O₂-Induced Death by Compound 3

The neuroprotective effect in SHSY5Y cells is Nurr1 dependent.Decreasing Nurr1 mRNA levels by 60% using a retrovirus caring shNurr1sequences (FIG. 2E), which decrease its transcriptional activity asassessed by luciferase assays (FIG. 2F), also decreases theneuroprotective effect of compound 3 treatment against MPP+ andincreases the sensitivity of the cells to this toxin (FIG. 2G).

VI. Single IP Administration of Compound 3 (20 mg/kg) Increased StriatalDA Levels and Dopamine Metabolite Levels in Wild Type C57/BL6 Mice

Single IP administration of the compound of the invention compound 3 (20mg/kg) increased striatal DA levels and dopamine metabolite (DOPAC andHVA) levels in wild type C57/BL6 mice by 33% (FIG. 3B). 4 hours afterdosing, animals were killed by cervical dislocation, brains wereremoved, and the striata were dissected on ice. The samples wereimmediately frozen and stored at −80° C. until use. The tissues wereprocessed for the analysis by high performance liquid chromatography(HPLC) with an electrochemical detector (ECD) (Emmanouil et al., 2006).The dissected tissues were weighed, homogenized, and deproteinized in0.2N perchioric acid solution containing 7.9 mM Na₂S₂O₅ and 1.3 mMNa₂EDTA. The homogenate was centrifuged at 37,000×g for 30 minutes, andthe supernatant was stored at −80° C. until assayed. A reverse-phaseion-pair chromatography was used in all analyses, and the mobile phaseconsisted of an acetonitrile-50 mM phosphate buffer at pH 3.0,containing 5-octylsulfate sodium salt (300 mg/L) as the ion-pair reagentand (20 mg/L) Na₂EDTA. Reference standards were prepared in 0.2 Nperchloric acid solution containing 7.9 mM Na₂S₂O₅ and 1.3 mM Na₂EDTA.Samples were quantified by comparison of the areas under the peaks withthose of reference standards using HPLC software (Chromatography Stationfor Windows™, Watrex International Inc., San Francisco, Calif.).

VII. Single IP Administration of Compound 3 (20 mg/kg) Increased THLevels in Wild Type C57/BL6 Mice

We measured striatal 5-HT levels, and we detected 5-HT and 5-HIM in wildtype C57/BL6 mice. 4 hours after dosing animals were killed by cervicaldislocation, brains were removed, and the striata were dissected on ice.The samples were immediately frozen and stored at −80° C. until use. Thetissues were processed for the analysis by high performance liquidchromatography (HPLC) with an electrochemical detector (ECD) (Emmanouilet al., 2006). The dissected tissues were weighed, homogenized, anddeproteinized in 0.2 N perchioric acid solution containing 7.9 mMNa₂S₂O₅ and 1.3 mM Na₂EDTA. The homogenate was centrifuged at 37,000×gfor 30 minutes, and the supernatant was stored at −80° C. until assayed.A reverse-phase ion-pair chromatography was used in all analyses, andthe mobile phase consisted of an acetonitrile-50 mM phosphate buffer atpH 3.0, containing 5-octylsulfate sodium salt (300 mg/L) as the ion-pairreagent and (20 mg/L) Na₂EDTA. Reference standards were prepared in 0.2N perchloric acid solution containing 7.9 mM Na₂S₂O₅ and 1.3 mM Na₂EDTA.Samples were quantified by comparison of the areas under the peaks withthose of reference standards using HPLC software (Chromatography Stationfor Windows™, Watrex International Inc., San Francisco, Calif.) (FIG.3C).

VIII. Single IP Administration of Compound 3 (20 mg/kg) Did not IncreaseNoradrenaline Levels in Wild Type C57/BL6 Mice

Single IP administration of the compound of the invention compound 3 (20mg/kg) did not increase noradrenaline levels in wild type C57/BL6 mice.4 hours after dosing animals were killed by cervical dislocation, brainswere removed, and the striata were dissected on ice. The samples wereimmediately frozen and stored at −80° C. until use. The tissues wereprocessed for the analysis by high performance liquid chromatography(HPLC) with an electrochemical detector (ECD) (Emmanouil et al., 2006).The dissected tissues were weighed, homogenized, and deproteinized in0.2 N perchloric acid solution containing 7.9 mM Na₂S₂O₅ and 1.3 mMNa₂EDTA. The homogenate was centrifuged at 37,000×g for 30 minutes, andthe supernatant was stored at −80° C. until assayed. A reverse-phaseion-pair chromatography was used in all analyses, and the mobile phaseconsisted of an acetonitrile-50 mM phosphate buffer at pH 3.0,containing 5-octylsulfate sodium salt (300 mg/L) as the ion-pair reagentand (20 mg/L) Na₂EDTA. Reference standards were prepared in 0.2 Nperchloric acid solution containing 7.9 mM Na₂S₂O₅ and 1.3 mM Na₂EDTA.Samples were quantified by comparison of the areas under the peaks withthose of reference standards using HPLC software (Chromatography Stationfor Windows™, Watrex International Inc., San Francisco, Calif.) (FIG.3D).

IX. Single IP Administration of Compound 3 (20 mg/kg) Increased MovementCoordination in Wild Type C57/BL6 PD Mice without Inducing Dyskinesia

Single IP administration of the compound of the invention compound 3 (20mg/kg) increased movement coordination in wild type C57/BL6 PD micetreated with MPTP. 20 mg/kg MPTP injections were administered 4 times atthe zero day of treatment spaced 2 hours apart. 7 days after the MPTPinjections there was approximately an 80% reduction in striatalprojections and 40%-50% dopaminergic neuron death in the SN. Motorcoordination and balance were tested using a Rota-Rod treadmill (UgoBasile, Comerio, Italy), set with accelerating revolution (4-40revolutions per min) over a 5 min period. Each mouse was placed on arotating drum, and the latency (in seconds) of each subject to fall fromthe rod was measured. Mice were given one training trial and 2experimental trials for 2 days (FIG. 4A).

Single IP administration of compound 3 (20 mg/kg) also increasedmovement coordination in wild type C57/BL6 PD mice treated with 6-OHDA.6-OHDA-HCl dissolved in PBS containing 0.02% ascorbic acid at aconcentration of 3.7 mg/ml was injected intracerebrally and unilaterallyat the level of MFB in anaesthetized mice. This regimen leads to 70%-80%dopaminergic cell death within 2 weeks after surgery. Motor coordinationand balance were tested using a Rota-Rod treadmill (Ugo Basile, Comerio,Italy), set with accelerating revolution (4-40 revolutions per min) overa 5 min period. Each mouse was placed on a rotating drum, and thelatency (in seconds) of each subject to fall from the rod was measured.Mice were given one training trial and 2 experimental trials for 2 days(FIG. 4B).

IP administration of compound 3 (20 mg/kg) repeated for 14 days did notinduce dyskinesias with abnormal involuntary movements (AIMS) in wildtype C57/BL6 PD mice treated with 6-OHDA. 6-OHDA-HCl dissolved in PBScontaining 0.02% ascorbic acid at a concentration of 3.7 mg/ml wasinjected intracerebrally and unilaterally at the level of MFB inanaesthetized mice. This regimen leads to 70%-80% dopaminergic celldeath within 2 weeks after surgery. 6-OHDA mice were left to recoverafter the surgery for 3 weeks. At that time, the degree of dopaminergicdegeneration was assessed for each mouse by the number of theapomorphine-induced turns it makes contralateral to the lesion. Thistest was used to create a relatively homogeneous group of mice withapproximately equal degrees of neurodegeneration. This group wassubsequently separated in 3 groups that were treated daily with an IPinjection of either saline, L-DOPA, or compound 3 (20 mg/kg) for 14days. At that time, abnormal involuntary movements were evaluated foreach of the 3 groups. Briefly, each mouse was observed individually for1 minute every 20 minutes for 3 hours, starting 20 minutes afterLDOPA/compound 3/saline administration. The AIMs were classified intothree different subtypes based on their topographic distribution: (i)axial AIMs, i.e., twisting of the neck and upper body towards the sidecontralateral to the lesion; (ii) orolingual AIMs, i.e., jaw movementsand contralateral tongue protrusion; (iii) forelimb AIMs, i.e.,purposeless movements of the contralateral forelimb. The mice wereevaluated using this test a total of 3 times during the chronic L-DOPAtreatment period. It was concluded that IP administration of thecompound of the invention compound 3 20 mg/kg) repeated for 12-17 daysdid not induce dyskinesias with abnormal involuntary movements (AIMs).In contrast, treatment of mice with L-DOPA, used as reference, induceddyskinesias with abnormal involuntary movements (AIMs) within 7 days(FIG. 4C).

X. Single IP Administration of Compound 3 (20 mg/kg) Increased ForcedSwim Test Performance in Wild Type C57/BL6 PD Mice

The chronic mild stress (CMS) procedure was applied semi randomly toeach mouse and consisted of a variety of unpredictable mildenvironmental, social and physical stressors, including confinement in asmall tube (1 hours), an empty cage without sawdust (15 hours), waterand food deprivation (15 hours), food restriction (approximately 50 mgof food pellets, 3 hours), cage tilted at 45° (1-3 hours), damp sawdust(approximately 200 ml of water per 100 g of sawdust, 3 hours), pairedhousing in damp sawdust (18 hours), reversal of the light/dark cycle,2-hour light/dark succession every 30 minutes, as well as light (15-17hours) and dark (4-6 hours). Compound 3 (2×10 mg/kg spaced 2 hoursapart) or vehicle control (Ethanol:PG:Saline, 10:30:60), wasadministered IP to mice. 4 hours after the last injection, mice wereevaluated in the Porsolt forced swim test. Mice aged 7-10 weeks wereplaced individually in a Plexiglas cylinder (diameter, 15 cm) containingwater (14-16 cm, 22-24.5° C.) The mice were videotaped for 5 min.Recorded behaviors for “floating” (the mouse is completely still in thewater, except for isolated movements to right itself) and “swimming”(movement of all four legs with body aligned horizontally in the water)were measured (in seconds) for each mouse. Administration of compound 3increased the swimming time by 31.7% and decreased the floating time by61.48% (FIG. 4D, 4E).

XI. IP Administration of Compound 3 (10 mg/kg) Protected Wild TypeC57/BL6 PD Mice from Midbrain Dopaminergic Neuronal Loss

IP administration of the compound of the invention compound 3 (10 mg/kg)repeated for 15 days every 12 hours protected wild type C57/BL6 PD micetreated with 6-OHDA from midbrain dopaminergic neuronal loss. 6-OHDA-HCldissolved in PBS containing 0.02% ascorbic acid at a concentration of3.7 mg/ml was injected intracerebrally and unilaterally at the level ofMFB in anaesthetized mice. This regimen leads to 70%-80% dopaminergiccell death within 2 weeks after surgery. 6-OHDA-treated mice were leftto recover after the surgery for 3 weeks. At that time, the degree ofdopaminergic degeneration was assessed for each mouse by the number ofthe apomorphine-induced turns it makes contralateral to the lesion. Thistest was used to create a relatively homogeneous group of mice withapproximately equal degrees of neurodegeneration. Mice wereanaesthetized deeply with CO₂ and perfused intracardially with ice-coldphosphate buffer (PBS; pH 7.2) and subsequently with 4% paraformaldehydein 0.1M PBS (PFA). Brains were quickly removed, post-fixed in PFAovernight at 4° C., cryoprotected in 15% sucrose in 0.1M PBS for 24hours and in 30% sucrose in 0.1M PBS for 24 hours at 4° C., frozen, andstored at −80° C. until sectioning. Free-floating cryostat-cut sections(30 μm) were collected using a Bright cryostat at −20° C. at the levelsof striatum (AP, 0.2 mm from bregma) and the entire midbrain (AP, −5.6mm from bregma) (Franklin and Paxinos, 2001). As previously described(Jackson-Lewis et al., 2000), sections first were quenched for 10minutes in 3% H₂O₂/10% methanol. Then sections were pre-incubated with10% goat serum for 1 hour and incubated with a polyclonal anti-THantibody (1:2,000; Calbiochem, San Diego, Calif.) for 48 h in 4° C.,followed by incubation with biotinylated anti-rabbit antibody (1:1500;Vector Laboratories, Burlingame, Calif.) in 1% goat serum for 1 hour andavidin-biotin peroxidase complex for 1 hour in RT (ABC Elite; VectorLaboratories). Staining was visualized using DAB (Sigma; St. Louis, Mo.)as a chromogen (Vila et al., 2000). The specificity was tested inadjacent sections with the primary or the secondary antibody omitted.The sections were stained with cresyl violet (Nissl staining) asdescribed previously (Franklin and Paxinos, 2001), and then dehydratedin graded ethanols and cover slipped. Total numbers of TH- andNissl-positive cells were counted in both hemi-brainstems by usingstereological methods (see, e.g., Jackson-Lewis et al., 2000). The totalnumber of TH-positive and Nissl-stained SNpc were counted by using theoptical fractionator, an unbiased method of cell counting that is notaffected by either the volume of reference or the size of the countedneurons. The SNpc was delineated by using a computer-assisted imageanalysis system (Jackson-Lewis et al., 2000). TH- and Nissl-stainedneurons were counted in every fourth section throughout the entireextent of the Snpc. The standard mouse atlas was used as an anatomicalreference (Franklin and Paxinos, 2001). Compound 3 treatment increasedthe number of surviving neuronal bodies by 256.55% (FIGS. 5A and 5B).

IP administration of compound 3 (10 mg/kg) repeated for 8 days every 12hours protected wild type C57/BL6 PD mice treated MPTP from midbraindopaminergic neuronal loss. 20 mg/kg MPTP injections were administered 4times at the zero day of treatment spaced 2 hours apart. 7 days afterthe MPTP injections there was approximately an 80% reduction in striatalprojections and 40%-50% dopaminergic neuron death in the SN. Mice wereanaesthetized deeply with CO₂ and perfused intracardially with ice-coldphosphate buffer (PBS; pH 7.2) and subsequently with 4% paraformaldehydein 0.1 M PBS (PFA). Brains were quickly removed, post-fixed in PFAovernight at 4° C., cryoprotected in 15% sucrose in 0.1 M PBS for 24hours and in 30% sucrose in 0.1 M PBS for 24 hours at 4° C., frozen, andstored at −80° C. until sectioning. Free-floating cryostat-cut sections(30 μm) were collected using a Bright cryostat at −20° C. at the levelsof striatum (AP, 0.2 mm from bregma) and the entire midbrain (AP, −5.6mm from bregma) (Franklin and Paxinos, 2001). As previously described(Jackson-Lewis et al., 2000), sections first were quenched for 10minutes in 3% H₂O₂/10% methanol. Then sections were pre-incubated with10% goat serum for 1 hour and incubated with a polyclonal anti-THantibody (1:2,000; Calbiochem, San Diego, Calif.) for 48 hours at 4° C.,followed by incubation with biotinylated anti-rabbit antibody (1:1500;Vector Laboratories, Burlingame, Calif.) in 1% goat serum for 1 hour andavidin-biotin peroxidase complex for 1 hour at room temperature (ABCElite; Vector Laboratories). Staining was visualized using DAB (Sigma;St. Louis, Mo.) as a chromogen (Vila et al., 2000). The specificity wastested in adjacent sections with the primary or the secondary antibodyomitted. The sections were stained with cresyl violet (Nissl staining)as described previously (Franklin and Paxinos, 2001), and thendehydrated in graded ethanols and cover slipped. Total numbers of TH-and Nissl-positive cells were counted in both hemi-brainstems by usingstereological methods (see, e.g., Jackson-Lewis et al., 2000). The totalnumber of TH-positive and Nissl-stained SNpc were counted by using theoptical fractionator, an unbiased method of cell counting that is notaffected by either the volume of reference or the size of the countedneurons. The SNpc was delineated by using a computer-assisted imageanalysis system (Jackson-Lewis et al., 2000). TH- and Nissl-stainedneurons were counted in every fourth section throughout the entireextent of the Snpc. T standard mouse atlas was used as an anatomicalreference (Franklin and Paxinos, 2001). Compound 3 treatment increasedthe number of surviving neuronal bodies by 33.5% (FIGS. 5 C and 5D).

IP administration of the compound of the invention compound 3 (10 mg/kg)every 12 hours for 8 days protected wild type 128/SV PD mice treatedwith MPTP from midbrain dopaminergic neuronal loss but not Nurr1heterozygote 129/SV mice treated with the same toxin. 20 mg/kg MPTPinjections were administered 4 times at the zero day of treatment,spaced 2 hours apart. 7 days after the MPTP injections there wasapproximately an 80% reduction in striatal projections and 40%-50%dopaminergic neuron death in the SN. Mice were anaesthetized deeply withCO₂ and perfused intracardially with ice-cold phosphate buffer (PBS; pH7.2) and subsequently with 4% paraformaldehyde in 0.1M PBS (PFA). Brainswere quickly removed, post-fixed in PFA overnight at 4° C.,cryoprotected in 15% sucrose in 0.1M PBS for 24 h and in 30% sucrose in0.1M PBS for 24 h at 4° C., frozen, and stored at −80° C. untilsectioning. Free-floating cryostat-cut sections (30p) were collectedusing a Bright cryostat at −20° C. at the levels of striatum (AP, 0.2 mmfrom bregma) and the entire midbrain (AP, −5.6 mm from bregma) (Franklinand Paxinos, 2001). As previously described (Jackson-Lewis et al.,2000), sections first were quenched for 10 min in 3% H₂O₂/10% methanol.Then sections were preincubated with 10% goat serum for 1 h andincubated with a polyclonal anti-TH antibody (1:2,000; Calbiochem, SanDiego, Calif.) for 48 h in 4° C., followed by incubation withbiotinylated anti-rabbit antibody (1:1500; Vector Laboratories,Burlingame, Calif.) in 1% goat serum for 1 h and avidin-biotinperoxidase complex for 1 h in RT (ABC Elite; Vector Laboratories).Staining was visualized using DAB (Sigma; St. Louis, Mo.) as a chromogen(Vila et al., 2000). The specificity was tested in adjacent sectionswith the primary or the secondary antibody omitted. The sections werestained with cresyl violet (Nissl staining) as described previously(Franklin and Paxinos, 2001), and then dehydrated in graded ethanols andcover slipped. Total numbers of TH- and Nissl-positive cells werecounted in both hemi-brainstems by using stereological methods (seebelow (Jackson-Lewis et al., 2000). The total number of TH-positive andNissl-stained SNpc were counted by using the optical fractionator, anunbiased method of cell counting that is not affected by either thevolume of reference or the size of the counted neurons. The SNpc wasdelineated by using a computer-assisted image analysis system(Jackson-Lewis et al., 2000). TH- and Nissl-stained neurons were countedin every fourth section throughout the entire extent of the Snpc. As ananatomical reference the standard mouse atlas was used (Franklin andPaxinos, 2001). Compound 3 treatment increased the number of survivingneuronal bodies by 33.5% in wild type 129/SV mice but did not lead to astatistically significant increase in surviving neuronal bodies in Nurr1heterozygote 129/SV mice (FIG. 5E).

XII. Bioavailability and Neuroprotective Effects of Compound 46

Compound 46 was found to be highly bioavailable and capable of crossingthe blood brain barrier. Administration by IP injection to mice revealedsignificant c-jun transcriptional activation in the midbrain asdetermined by qPCR 2 hours after administration (FIG. 70A).

Additionally, experiments were conducted to assess whether compound 46possesses neuroprotective properties. Such assays were performed, forinstance, by adding compound 46 (12.5 μM) to human origin SHSY-5Ydopaminergic cells in which death was induced by the mitochondriacomplex I inhibitor MPP+(1-methyl-4-phenylpyridinium), the activemetabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).Pretreatment of the cells with compound 46 for 12 to 24 hours prior toincubation with MPP+significantly increased the survival of cellsagainst varying concentrations of the toxic stimulus. Most of theMPP+-treated cells receiving vehicle died, while the few surviving cellsremained attached to the plate but were rounded and had lost allprojections. These morphological changes indicated impaired neuronalfunction. On the contrary, cells treated with compound 46 appearedhealthy, flattened, well-attached, and their projections remainedintact. The relative survival of SHSY-5Y dopaminergic cells treated withand without compound 46 is shown in FIG. 70B.

XIII. Summary of Biological Activity of Compound 3

Parkinson's disease (PD) is a progressive neurodegenerative disordercharacterized by the loss of dopaminergic neurons in the substantianigra and the gradual depletion of dopamine. Current treatments aim toreplenish the dopamine deficit and to improve symptoms, but, over time,they induce dyskinesias while neuroprotective therapies are nonexistent.Here, we report that Nurr1:RXRα activation has a double therapeuticpotential for PD, offering both neuroprotective and symptomaticimprovement. We designed compound 3, a unique in vivo activeNurr1:RXRα-selective lead small molecule, which prevented dopaminergicneuron demise against PD-causing toxins and PD-related geneticmutations, in a Nurr1-dependent manner, in both patient iPSc-deriveddopaminergic neurons and in preclinical mouse PD models. Compound 3 invivo maintained striatal dopaminergic innervation and alleviated motorsymptoms. Remarkably, besides neuroprotection, compound 3 upregulatedTH, AADC and GCH1 transcription, increased striatal dopamine in vivo andhad symptomatic efficacy in two post-neurodegeneration PD models,without inducing dyskinesias upon chronic daily treatment. The combinedneuroprotective and symptomatic effects of compound 3 designatesNurr1:RXRα activation as a potential monotherapy approach in PD.

XCT0135908 In Vivo: Low Stability and Low Brain Penetration

XCT0135908 was administered to mice to test its bioactivity.Intraperitoneal (IP) or intracerebroventricular XCT0135908 (1 and 10mg/kg) injections did not result in any expression alterations ofmidbrain genes such c-jun or TH at different time points afteradministration. LC-MS/MS analysis of blood plasma or brain homogenatesand targeted search of the parent compound 1 and 2 hours after IPXCT0135908 (1 μg/kg) administration indicated low compound stability andminimal brain penetration (brain/blood<0.03) (FIGS. 68A-B).

Nurr1:RXRα Activator Compound 3

In silico docking simulations show compound 3 to complement thehydrophobic L-shaped binding pocket of RXRα (pdb1MV9). The carboxylicgroup of compound 3, with the participation of a water molecule, forms ahydrogen bond with the backbone amide of A327 and a salt bridge with theside chain of R316. The phenyl ring of compound 3 participates in π-πinteractions with F346 and stabilizes the molecule in the RXRα bindingpocket contributing to target affinity (calculated ΔG −16.7 kcal/mol)(FIG. 71). These simulations were validated by methyl or ethyl estercarboxylic group modification, which abolished activation, increasingEC50 by 50 to 100 fold. In addition, halogenation of the compound 3phenyl ring disturbs the π-π interactions with F346 and also increasesthe EC50 by approximately 7-fold.

Specificity of Compound 3 for Nurr1:RXRα Heterodimers and BrainPenetration

Compound 3 activated Nurr1:RXRα heterodimers (FIG. 1C) but did notactivate Nurr1:RXRγ heterodimers (FIG. 1D), indicating specificity forRXRα and that it does not bind to Nurr1. In naïve SHSY-5Y cells,compound 3 activated endogenous Nurr1:RXRα heterodimers, as verified byloss of activity after knocking-down Nurr1 by approximately 60% using aretrovirus carrying shNurr1 sequences (FIG. 2E) indicating that Nurr1 isrequired for compound 3 activity (FIG. 2F).

To test off-target effects of compound 3 on complexes aside fromNurr1:RXRα heterodimers, we fused the ligand binding domains of Nurr1,RXRα and a variety of related nuclear receptors to GAL4 DNA-bindingdomain to create chimeric proteins, while the ligand-binding domain ofRXRα was also fused with VP16. These molecular chimeras wereco-transfected in pairs with the RXRα:VP16 along with a GAL4-responsiveluciferase reporter in SHSY-5Y, which were stimulated with compound 3.Compound 3 strongly activated Nurr1GAL4:RXRαVP16 heterodimer chimerasbut failed to activate other RXRαVP16 heterodimer chimeras with VDRGAL4,RXRγGAL4, PPARγGAL4 as well as RXRαGAL4 homodimer chimeras (FIG. 1E).Nur77GAL4:RXRαVP16 heterodimers were partially activated but, Nur77,unlike Nurr1, is not associated with PD and Nur77 knock-down enhancescell survival (Wei et al. Mol Neurobiol. doi:10.1007/s12035-015-9477-7,2015). The above experiments indicate the high degree of selectivity ofcompound 3 and point to its neuroprotective potential.

IP administration of compound 3 (1 mg/kg) in mice resulted in compound 3reaching the brain. This compound exhibited an approximate half-life ofabout 2 hours in both blood and brain as assessed by LC-MS/MS(brain/blood concentration AUC ratio 1.7, FIGS. 69A-B) and wasbioactive, as it increased midbrain c-jun expression (FIG. 69C).

Compound 3 Induces Transcription of DA Biosynthesis Genes and ProtectsDAergic Cell Lines and Human iPSC Derived DAergic Neurons Against PDAssociated Damage

Nurr1 regulates the transcription of DA biosynthesis genes (Kim et al.J. Neurochem. 85:622-634, 2003 and Gil et al. J Neurochem 101:142-150,2007); however, whether this process is mediated by Nurr1:RXRαheterodimers is unknown. In the human DAergic cell line SHSY-5Y,compound 3, but not vehicle, increased the expression of the three genesrequired for DA biosynthesis, TH by about 90% (n=6, t-test p=0.0374),aromatic L-amino acid decarboxylase (MDC) by about 70% (n=6, t-testp<0.0001), and GCH1 by about 42% (n=6, t-test p<0.0001), (FIG. 72A),indicating that this up-regulation depends upon Nurr1:RXRα heterodimeractivation.

We examined the neuroprotective capability of Nurr1:RXRα heterodimeractivation in SHSY-5Y cells where death was induced by either theoxidative stress related H₂O₂ or the mitochondria complex I inhibitorMPP+(1-methyl-4-phenylpyridinium) (FIG. 72B). Compound 3 (12.5 μM)significantly increased cell survival against varying concentrations ofthe toxic stimuli (2-way ANOVA P<0.0001) in a compound 3 dose-dependentmanner (FIG. 72C). The protection conferred by compound 3 wasNurr1-dependent, since it was abrogated by knocking-down endogenousNurr1 mRNA (FIGS. 1E and 72D). The neuroprotective effects of compound 3extend to damage induced by the PD-associated mutation G2019S in theLeucine-Rich Repeat Kinase 2 (LRRK2) gene, the most common geneticdefect associated with clinical PD (Tofaris et al. J. Neurosci.26:3942-3950, 2006). Rat cortical neurons, co-transfected with CMV-GFPfor identification, and CMVLRRK2-G2019S cDNAs show increased apoptosisas assessed by DAPI staining, compared to control neurons co-transfectedwith a LRRK2 wt cDNA. Compound 3 treatment reduced apoptosis to controllevels, comparable to those the LRRK2 inhibitor GSK2578215A (1-way ANOVAP<0.0001) (FIGS. 72E-72I).

To ascertain the translational therapeutic potential of compound 3, wetested whether human midbrain-specific iPSc-derived DAergic neurons, asindicated by positive staining and qPCR for the neuronal markers MAP2,TH, FoxA2, Lmx1 and Ent, can be protected from MPP+ toxicity. Compound 3was able to double the number of surviving neurons after MPP+ exposurewithin 24 hours, compared to vehicle (FIGS. 72J-72L). The survivingcontrol neurons had fewer and retreating projections, indicatingcompromised function (FIG. 72K) while the neurons that had receivedcompound 3 had retained a complex network of projections and contacts(FIG. 72L). In addition, we tested compound 3 in iPSc-derived DAergicneurons from a PD patient carrying the LRRK2-G2019S mutation. Theseneurons showed contracted neurites with reduced branching, phenomenathat can be reversed upon correction (LRRK2GC) of the LRRK2-G2019Smutation (Tofaris et al. J. Neurosci. 26:3942-3950, 2006). Treatment ofthe LRRK2-G2019S mutated DAergic neurons with compound 3 (12.5 μM) for14 days (FIGS. 72M-72Q) also increased neurite length, number andbranching by 83% (1-way ANOVA p<0.0007; Kruskal-Wallis p<0.01), 38%(1-way ANOVA p<0.0001; Kruskal-Wallis p<0.001) and 650% respectively(1-way ANOVA p<0.01; Kruskal-Wallis p<0.05) (FIGS. 72M-72O).

These experiments demonstrate the in vitro potential of Nurr1:RXRαactivation to shield human DAergic neurons from diverse PD relatedneuronal death stimuli such as toxins and the LRRK2-G2019S mutation.

Neuroprotection of Compound 3 In Vivo in Preclinical Mouse PD Models

We assessed the neuroprotective effects of Nurr1:RXRα heterodimeractivation in vivo in two established preclinical C57BL/6 mouse PDmodels: the acute MPTP and the unilateral 6-OHDA injection in the SN.Compound 3 IP injections (10 mg/kg) every 12 hours, starting 12 hoursbefore toxin administration, were continued for 6 days (MPTP) and 14days (6-OHDA). To distinguish phenotypic effects, lasting 4-8 hours,from neuroprotection, compound 3 administration was discontinued 24-36hours before behavioral and histological examination of the mice. In theMPTP model, mice receiving compound 3 had considerably improved motorcoordination (>100%) (1 way ANOVA, P<0.0001) than vehicle-injected mice,as assessed by the accelerating rotarod (FIG. 73A). SN unbiasedstereological neuronal counting showed that TH(+) midbrain neuronsurvival was increased by 31% (1 way ANOVA, p=0.0003) in compound3-treated animals, to a value not significantly different from controlmice (FIGS. 73B-C). Additionally, compound 3 protected SN axonalprojections to the striatum, doubling the number of remaining terminals(1 way ANOVA, P<0.0001) (FIGS. 73D-E). The neuroprotection was compound3 dependent because a once-a-day injection regimen (20 mg/kg) wasineffective. Compound 3 neuroprotection against MPTP toxicity wasequally effective in 129sv wt mice (1 way ANOVA, P<0.001), but it wasabolished in Nurr1+/−129sv animals, validating that the in vivoneuroprotective effects of compound 3 require Nurr1:RXRα heterodimers(FIG. 73F).

Nurr1:RXRα activation showed even more striking effects in the 6-OHDAmodel. Compound 3 produced a dramatic reduction of apomorphine inducedcontralateral turns by approximately 8-fold (Mann Whitney, p=0.0159)(FIG. 74A), while motor coordination was almost entirely restored tocontrol levels, as evaluated by the rotarod test (1 way ANOVA, P<0.0001,Mann Whitney v0.0079) (FIG. 74B). The number of surviving TH(+) neuronsin the SN of compound 3-treated mice was increased by 47%, (1 way ANOVA,P<0.05, Mann Whitney, p=0.0317) to the level of complete restoration, asindicated by unbiased stereological neuronal counting (FIGS. 74C-D).Total neuronal determination by NueN staining showed similar results.Striatal innervation, which was practically obliterated in thevehicle-treated control mice, also showed a 10-fold higher preservationof TH(+) DAergic projections (1 way ANOVA p<0.0001, Mann Whitneyp=0.0159) (FIGS. 74E-F).

Because the predictive validity of toxin-based mouse PD models forneuroprotection in humans is questionable, we proceeded to test theeffect of compound 3 in a mouse model, where AAV viruses overexpressingwt alpha-synuclein (AAV-ASYN) under the chicken beta-actin promoter wereunilaterally injected, combined with contralateral injections of(AAV-GFP). The same compound 3 (10 mg/kg every 12 h for 2 weeks) orvehicle treatment regimen was followed as in the toxin models. Unbiasedstereology showed that the vehicle-treated AAV ASYN injected animalssuffered a 44% decrease of TH(+) and NeuN(+) midbrain neurons incomparison to the AAV-GFP injected side (FIGS. 74G-I). On the contrary,in compound 3-treated AAV-ASYN-injected animals, unbiased stereologyshowed that the number of NeuN(+) midbrain neurons increased (Wilcoxonp<0.05; Mann-Whitney p=0.00214) and the quantity of TH(+) midbrainneurons was increased by about 47% (Wilcoxon p<0.05; Mann-Whitneyp=0.00214) in comparison to AAV-ASYN injected animals treated withvehicle (FIG. 74G). Striatal innervation of unilaterally AAV-ASYNinjected brains resulted in a dramatic depletion of TH(+) axons byalmost 85% in animals treated with vehicle, wheareas in compound3-treated animals striatal TH(+) axons were increased over 5 fold (1 wayANOVA, P<0.001, Wilcoxon p<0.0156)(FIGS. 74J-L).

These experiments demonstrate DAergic neuroprotective effects ofNurr1:RXRα activation in both toxin-based and genetic preclinical animalmodels of PD.

Chronic Daily Treatment with Compound 3 Induces DA Biosynthesis In Vivoand Symptomatic Efficacy without Dyskinesias in TwoPost-Neurodegeneration PD Models

Given the role of Nurr1:RXRα activation in in the transcriptionalregulation of DA biosynthesis genes, we tested whether compound 3 couldincrease DA levels in wt and ASYN transgenic mice (Tofaris et al. J.Neurosci. 26:3942-3950, 2006), which display reduced striatal DA levelsafter reaching adulthood. A single compound 3 (10 mg/kg) IP injection inwt mice resulted in increased TH gene expression (t test p=0.0396) inthe midbrain within 4 hours (FIG. 75A). Striatal DA and DA metabolitelevels were also increased, and their ratio remained constant,indicating physiological DA catabolism (FIG. 75B). Aiming to model PDbased on human genetic data, we also tested whether compound 3 couldincrease DA levels in ASYN transgenic mice. A single IP injection ofcompound 3 (10 mg/kg) was able increase striatal DA (t test p=0.01069)and DA metabolite levels in ASYN transgenics as well (FIG. 75C) incomparison to mice receiving vehicle. The effect of compound 3 waslimited to DA biosynthesis, since noradrenaline levels remainedunaffected (FIG. 75D).

We subsequently tested whether the DA increase could be translated insymptomatic relief in post-degeneration PD mouse models based ontreatment with MPTP or 6-OHDA (FIGS. 75E-F). A single dose of compound 3(10 mg/kg) 8 days post-acute MPTP injection or 5-6 weeks post-unilateral6-OHDA injection, significantly improved temporarily motor coordinationin both models (1-way ANOVA, P=0.01) (1-way ANOVA, P=0.0001)respectively 4 hours, post-dosing (FIGS. 75G-H) and inducedcontralateral turns in the 6-OHDA model (1-way ANOVA, P<0.0001) (FIG.75I), an effect similar to that of levodopa. Compound 3-inducedsymptomatic improvement in these models disappeared 8 hours post-dosing.These experiments indicate that Nurr1:RXRα activation can indeed offersymptomatic relief in rodents, further validating our monotherapyhypothesis.

Significantly, compound 3 was compared to levodopa in causing AIMs. Micetreated daily with levodopa, starting 21 days post-unilateral 6-OHDAinjection, displayed severe hyperkinetic dyskinesias/AIMs within 7 days(FIGS. 4C and 75J). In contrast, similar daily compound 3 (10 mg/kg)administration for at least two weeks resulted in consistently improvedmotor coordination (FIGS. 75H-I) without resulting in dyskinesias/AIMs(2-way ANOVA, p=0.0061, Mann Whitney p=0.0043) (FIG. 4C). Theseexperiments exemplify the simultaneous symptomatic usefulness ofNurr1:RXRα activation devoid of undesirable complications.

REFERENCES

-   Anderson G, Noorian A R, Taylor G, Anitha M, Bernhard D, Srinivasan    S, Greene J G. Loss of enteric dopaminergic neurons and associated    changes in colon motility in an MPTP mouse model of Parkinson's    disease. Exp Neurol. 2007 September; 207(1):4-12.-   Arfaoui A, Lobo M V, Boulbaroud S, Ouichou A, Mesfioui A, Arenas    M I. Expression of retinoic acid receptors and retinoid X receptors    in normal and vitamin A deficient adult rat brain. Ann Anat. 2012.    pii: S0940-9602(12)00129-X.-   Backman C, Perlmann T, Wallen A, Hoffer B J, Morales M. A selective    group of dopaminergic neurons express Nurr1 in the adult mouse    brain. Brain Res. 1999:851(1-2):125-32.-   Berrios, G E (1985). “The Psychopathology of Affectivity: Conceptual    and Historical Aspects”. Psychological Medicine 15 (4): 745-758.    doi:10.1017/S0033291700004980.PMID 3909185.-   Buervenich S, Carmine A, Arvidsson M, Xiang F, Zhang Z, Sydow O,    Jönsson E G, Sedvall G C, Leonard S, Ross R G, Freedman R, Chowdari    K V, Nimgaonkar V L, Perlmann T, Anvret M, Olson L. NURR1 mutations    in cases of schizophrenia and manic-depressive disorder. AJMG. 2000;    96(6):808-13.-   Carlson, C. Donald; Heth (2007). Psychology the science of behavior    (4th ed.). Pearson Education Inc. ISBN 0-205-64524-0-   Chaudhuri K R, Schapira A H. Non-motor symptoms of Parkinson's    disease: dopaminergic pathophysiology and treatment. Lancet Neurol.    2009; 8(5):464-74.-   Chu Y, Kompoliti K, Cochran E J, Mufson E J, Kordower J H.    Age-related decreases in Nurr1 immunoreactivity in the human    substantia nigra. J Comp Neurol. 2002 Aug. 26; 450(3):203-14.-   Chu Y, Kordower J H. Age-associated increases of alpha-synuclein in    monkeys and humans are associated with nigrostriatal dopamine    depletion: Is this the target for Parkinson's disease? Neurobiol    Dis. 2007 January; 25(1):134-49.-   Emmanouil D E, Papadopoulou-Daifoti Z, Hagihara P T, Quock D G,    Quock R M. A study of the role of serotonin in the anxiolytic effect    of nitrous oxide in rodents. Pharmacol Biochem Behav. 2006 June;    84(2):313-20. Epub 2006 Jul. 7-   Franklin K B J, Paxinos G. The Mouse Brain in Stereotaxic    Coordinates. San Diego: Academic Press; 2001.-   Grimes D A, Han F, Panisset M, Racacho L, Xiao F, Zou R, Westaff K,    Bulman D E. Translated mutation in the Nurr1 gene as a cause for    Parkinson's disease. Mov Disord. 2006 July; 21(7):906-9.-   Henchcliffe C, Severt W L. Disease modification in Parkinson's    disease. Drugs Aging. 2011 Aug. 1; 28(8):605-15-   Hering R, Petrovic S, Mietz E M, Holzmann C, Berg D, Bauer P,    Woitalla D, Muller T, Berger K, Krüger R, Riess O. Extended mutation    analysis and association studies of Nurr1 (NR4A2) in Parkinson    disease. Neurology. 2004 Apr. 13; 62(7):1231-2.-   Healy D G, Abou-Sleiman P M, Ahmadi K R, Gandhi S, Muqit M M, Bhatia    K P, Quinn N P, Lees A J, Holton J L, Revesz T, Wood N W. NR4A2    genetic variation in sporadic Parkinson's disease: a genewide    approach. Mov Disord. 2006 November; 21(11):1960-3.-   Hermanson E, Borgius L, Bergsland M, Joodmardi E, Perlmann T.    Neuropilin1 is a direct downstream target of Nurr1 in the developing    brain stem. J Neurochem. 2006 June; 97(5):1403-11-   Jackson-Lewis V, Liberatore G. Effects of a unilateral stereotaxic    injection of Tinuvin 123 into the substantia nigra on the    nigrostriatal dopaminergic pathway in the rat. Brain Res. 2000 Jun.    2; 866(1-2):197-210.-   Jacobsen K X, MacDonald H, Lemonde S, Daigle M, Grimes D A, Bulman D    E, Albert P R A Nurr1 point mutant, implicated in Parkinson's    disease, uncouples ERK1/2-dependent regulation of tyrosine    hydroxylase transcription. Neurobiol Dis. 2008 January;    29(1):117-22.-   Le W D, Xu P, Jankovic J, Jiang H, Appel S H, Smith R G, Vassilatis    D K. Mutations in NR4A2 associated with familial Parkinson disease.    Nat Genet. 2003 January; 33(1):85-9.-   Lewis, A J (1934). “Melancholia: A Historical Review.”. Journal of    Mental Science 80(328): 1-42. doi:10.1192/bjp.80.328.1.-   Li Z S, Schmauss C, Cuenca A, Ratcliffe E, Gershon M D.    Physiological modulation of intestinal motility by enteric    dopaminergic neurons and the D2 receptor: analysis of dopamine    receptor expression, location, development, and function in    wild-type and knock-out mice. J Neurosci.2006; 26:2798-807.-   Luo Y, Henricksen L A, Giuliano R E, Prifti L, Callahan L M,    Federoff H J. VIP is a transcriptional target of Nurr1 in    dopaminergic cells. Exp Neurol. 2007 January; 203(1):221-32.-   McDowell K, Chesselet M F. Animal models of the non-motor features    of Parkinson's disease. Neurobiol Dis. 2012 June; 46(3):597-606.-   Monaca C, Laloux C, Jacquesson J M, Gelé P, Marechal X, Bordet R,    Destee A, Derambure P. Vigilance states in a parkinsonian model, the    MPTP mouse. Eur J Neurosci. 2004; 20(9):2474-8.-   Pack A I, Galante R J, Maislin G, Cater J, Metaxas D, Lu S, Zhang L,    Von Smith R, Kay T, Lian J, Svenson K, Peters L L.Novel method for    high-throughput phenotyping of sleep in mice.Physiol Genomics. 2007;    28(2):232-8.-   Prashanth L K, Fox S, Meissner W G. I-Dopa-induced    dyskinesia-clinical presentation, genetics, and treatment. Int Rev    Neurobiol. 2011; 98:31-54.-   Rosland J H, Hunskaar S, Broch O J, Hole K. Acute and long term    effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in    tests of nociception in mice. Pharmacol Toxicol. 1992 January;    70(1):31-7.-   Santiago R M, Barbieiro J, Lima M M, Dombrowski P A, Andreatini R,    Vital M A. Depressive-like behaviors alterations induced by    intranigral MPTP, 6-OHDA, LPS and rotenone models of Parkinson's    disease are predominantly associated with serotonin and dopamine.    Prog Neuropsychopharmacol Biol Psychiatry. 2010 Aug. 16;    34(6):1104-14-   Sleiman P M, Healy D G, Muqit M M, Yang Y X, Van Der Brug M, Holton    J L, Revesz T, Quinn N P, Bhatia K, Diss J K, Lees A J, Cookson M R,    Latchman D S, Wood N W. Characterisation of a novel NR4A2 mutation    in Parkinson's disease brain. Neurosci Lett. 2009 Jun. 26;    457(2):75-9.-   Sousa K M, Mira H, Hall A C, Jansson-Sjöstrand L, Kusakabe M,    Arenas E. Microarray analyses support a role for Nurr1 in resistance    to oxidative stress and neuronal differentiation in neural stem    cells. Stem Cells. 2007 February; 25(2):511-9.-   Tadaiesky M T, Dombrowski P A, Figueiredo C P, Cargnin-Ferreira E,    Da Cunha C, Takahashi R N. Emotional, cognitive and neurochemical    alterations in a premotor stage model of Parkinson's disease.    Neuroscience. 2008 Oct. 28; 156(4):830-40.-   Tofaris G K, Garcia Reitböck P, Humby T, Lambourne S L, O'Connell M,    Ghetti B, Gossage H, Emson P C, Wilkinson L S, Goedert M,    Spillantini M G. Pathological changes in dopaminergic nerve cells of    the substantia nigra and olfactory bulb in mice transgenic for    truncated human alpha-synuclein(1-120): implications for Lewy body    disorders. J Neurosci. 2006; 26(15):3942-50.-   Vila M, Vukosavic S, Jackson-Lewis V, Neystat M, Jakowec M,    Przedborski S.Alpha-synuclein up-regulation in substantia nigra    dopaminergic neurons following administration of the parkinsonian    toxin MPTP J Neurochem. 2000 February; 74(2):721-9.-   Volpicelli F, Caiazzo M, Greco D, Consales C, Leone L,    Perrone-Capano C, Colucci D'Amato L, di Porzio U. Bdnf gene is a    downstream target of Nurr1 transcription factor in rat midbrain    neurons in vitro. J Neurochem. 2007 July; 102(2):441-53.-   Wallen-Mackenzie A, Mata de Urquiza A, Petersson S, Rodriguez F J,    Friling S, Wagner J, Ordentlich P, Lengqvist J, Heyman R A, Arenas    E, Perlmann T. Nurr1-RXR heterodimers mediate RXR ligand-induced    signaling in neuronal cells.Genes Dev. 2003 Dec. 15; 17(24):3036-47.-   Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker    N P, Perlmann T.Structure and function of Nurr1 identifies a class    of ligand-independent nuclear receptors. Nature. 2003 May 29;    423(6939):555-60.-   Xiao Q, Castillo S O, Nikodem V M.Distribution of messenger RNAs for    the orphan nuclear receptors Nurr1 and Nur77 in adult rat brain    using in situ hybridization. Neuroscience. 1996; 75(1):221-30-   Xilouri M, Kyratzi E, Pitychoutis P M, Papadopoulou-Daifoti Z,    Perier C, Vila M, Maniati M, Ulusoy A, Kirik D, Park D S, Wada K,    Stefanis L. Selective neuroprotective effects of the S18Y    polymorphic variant of UCH-L1 in the dopaminergic system. Hum Mol    Genet. 2012; 21(4):874-89-   Arfaoui A, Lobo M V, Boulbaroud S, Ouichou A, Mesfioui A, Arenas    M I. Expression of retinoic acid receptors and retinoid X receptors    in normal and vitamin A deficient adult rat brain. Ann Anat. 2012.    pii: S0940-9602(12)00129-X.-   Bäckman C, Perlmann T, Wall& A, Hoffer B J, Morales M. A selective    group of dopaminergic neurons express Nurr1 in the adult mouse    brain. Brain Res. 1999:851(1-2):125-32.-   Decressac M, Volakakis N, Bjorklund A, Perlmann T.NURR1 in Parkinson    disease—from pathogenesis to therapeutic potential. Nat Rev Neurol.    2013 November; 9(11):629-36.-   Franklin K B J, Paxinos G. The Mouse Brain in Stereotaxic    Coordinates. San Diego: Academic Press; 2001.-   Henchcliffe C, Severt W L. Disease modification in Parkinson's    disease. Drugs Aging. 2011 Aug. 1; 28(8):605-15-   Overington JP¹, Al-Lazikani B, Hopkins A L. How many drug targets    are there? Nat Rev Drug Discov. 2006 December; 5(12):993-6.-   Pérez E¹, Bourguet W, Gronemeyer H, de Lera A R. Modulation of RXR    function through ligand design. Biochim Biophys Acta. 2012 January;    1821(1):57-69.-   Rosland J H, Hunskaar S, Broch O J, Hole K. Acute and long term    effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in    tests of nociception in mice. Pharmacol Toxicol. 1992 January;    70(1):31-7.-   Vaz B¹, de Lera A R. Advances in drug design with RXR modulators.    Expert Opin Drug Discov. 2012 Noemberv; 7(11):1003-16.-   Vila M, Vukosavic S, Jackson-Lewis V, Neystat M, Jakowec M,    Przedborski S. Alpha-synuclein up-regulation in substantia nigra    dopaminergic neurons following administration of the parkinsonian    toxin MPTP J Neurochem. 2000 February; 74(2):721-9.-   Wallen-Mackenzie A, Mata de Urquiza A, Petersson S, Rodriguez F J,    Friling S, Wagner J, Ordentlich P, Lengqvist J, Heyman R A, Arenas    E, Perlmann T. Nurr1-RXR heterodimers mediate RXR ligand-induced    signaling in neuronal cells.Genes Dev. 2003 Dec. 15; 17(24):3036-47.-   Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker    N P, Perlmann T. Structure and function of Nurr1 identifies a class    of ligand-independent nuclear receptors. Nature. 2003 May 29;    423(6939):555-60.

1.-9. (canceled)
 10. A compound of formula 1e:

or a pharmaceutically acceptable salt thereof, wherein n is 0 to 2, p is0 to 2; each R₁ is independently selected from the group consisting ofhalogen, cyanate, isocyanate, thiocyanate, isothiocyanate,selenocyanate, isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano,nitro, hydroxy, acyl, mercapto, carboxyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR₇,—SR₇, and —N(R₈)R₇; each R₂ is independently selected from the groupconsisting of halogen, cyanate, isocyanate, thiocyanate, isothiocyanate,selenocyanate, isoselenocyanate, alkoxy, trifluoromethoxy, azido, cyano,nitro, hydroxy, acyl, mercapto, carboxyl, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR₇,—SR₇, and —N(R₈)R₇; R₄ is selected from the group consisting ofhydrogen, halogen, optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedaryl-alkyl, optionally substituted heterocyclyl, optionally substitutedheteroaryl, optionally substituted heteroaryl-alkyl, optionallysubstituted heterocyclolalkyl, OR₇, and —N(R₈)R₇; R₅ is selected fromthe group consisting of hydrogen, optionally substituted alkyl,optionally substituted alkenyl, and optionally substituted aryl; each R₇is independently selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted aryl, optionally substituted aryl-alkyl, and optionallysubstituted heterocyclyl; and each R₈ is independently selected from thegroup consisting of hydrogen and optionally substituted alkyl; R₁₀ isselected from the group consisting of hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aryl-alkyl,optionally substituted heteroaryl, optionally substituted cycloalkyl,and optionally substituted heterocyclyl; and R₁₁ is selected from thegroup consisting of optionally substituted alkyl, optionally substitutedaryl, optionally substituted aryl-alkyl, optionally substitutedheteroaryl, optionally substituted cycloalkyl, and optionallysubstituted heterocyclyl.
 11. The compound of claim 10, wherein R₄ ismethyl or trifluoromethyl.
 12. The compound of claim 10, wherein R₅ ishydrogen or alkyl.
 13. The compound of claim 10, wherein n is 0 or 1,and wherein p is 0 or
 1. 14. The compound of claim 10, wherein R₄ ishaloalkyl.
 15. The compound of claim 10, wherein R₄ is trifluoromethyl.16. A compound of claim 10 selected from the group consisting of:

o) or a salt or free base thereof.
 17. A pharmaceutical compositioncomprising a compound of claim 10 and a pharmaceutically acceptableexcipient.
 18. The pharmaceutical composition of claim 17, wherein saidpharmaceutical composition comprises an additional therapeuticallyactive compound.
 19. The pharmaceutical composition of claim 18, whereinsaid additional therapeutically active compound is selected from thegroup consisting of levodopa (L-dihydroxyphenylalanine), L-aromaticamino acid decarboxylase (AADC) inhibitors, and catechol O-methyltransferase (COMT) inhibitors.
 20. A method of treating aneurodegenerative disorder in a human, or alleviating or preventing oneor more symptoms of a neurodegenerative disorder in a human, said methodcomprising administering to the human an effective amount of a compoundof claim
 10. 21. (canceled)
 22. The method of claim 20, wherein saidsymptom is selected from the group consisting of bradykinesia anddyskinesias.
 23. The method of claim 20, wherein said symptom isselected from the group consisting of anxiety and depression.
 24. Themethod of claim 20, wherein said symptom is selected from the groupconsisting of learning difficulties, memory disorders, and attentiondeficit disorder.
 25. The method of claim 20, wherein said symptom isselected from the group consisting of insomnia and katalepsy. 26.(canceled)
 27. The method of claim 20, wherein said disorder isParkinson's disease.
 28. The method of claim 20, wherein the effectiveamount is sufficient to halt or prevent degeneration of neurons.
 29. Themethod of claim 28, wherein said neurons are dopaminergic midbrainneurons.
 30. The method of claim 20, wherein the effective amount issufficient to increase the rate of dopamine biosynthesis. 31.-48.(canceled)